Therapeutic

The invention relates to recombinant polynucleotides, compositions and methods for interfering with antibiotic resistance genes, and/or replicons carrying such genes, in microorganisms in order to disable antibiotic resistance in the microorganisms.

Antibiotics, originally isolated from microorganisms such as Streptomyces, are a powerful way to treat infectious disease. However, very quickly bacteria acquired anti-microbial resistance (AMR) to antibiotics in response to selection pressure. One common route to AMR has been the acquisition of resistance genes evolved in the original antibiotic-producing microorganisms, via horizontal transmission on plasmid vectors. Such plasmids have in some instances acquired multiple antibiotic resistance genes carried by transposable elements and integrons. Host-encoded mutations that modify the bacterial protein target or prevent entry of the antibiotic have also occurred.

Resistance to antibiotics by microorganisms such as bacterial pathogens is one of our most serious health threats. Infections from resistant bacteria, for example, are now not uncommon, and some pathogens have even become resistant to multiple types or classes of antibiotics. The loss of effective antibiotics undermines our ability to fight infectious diseases and manage the infectious complications common in vulnerable patients, for example those undergoing chemotherapy for cancer, dialysis for renal failure, and surgery, especially organ transplantation, for which the ability to treat secondary infections is critical.

When first-line and second-line treatment options are limited by antibiotic resistance or are unavailable, healthcare providers are forced to use alternative antibiotics that may be more toxic to the patient, more expensive and less effective. Even when alternative treatments exist, patients with resistant infections are often more likely to die, while survivors may have significantly longer hospital stays, delayed recuperation, and long-term disability. Many achievements of modern medicine are put at risk by AMR. Without effective antibiotics for care and prevention of infections, the success of treatments such as organ

transplantation, cancer chemotherapy and major surgery would be compromised.

The growth of global trade and travel allows antibiotic resistant microorganisms to be spread rapidly through humans, other animals, and food.

Resistance mechanisms fall into four classes: (1 ) enzymes that degrade antibiotics, including beta-lactamases that break the beta-lactam ring of the penicillin family of antibiotics;

(2) enzymes that modify antibiotics include aminoglycoside phosphotransferases that phosphorylate aminoglycoside antibiotics such as kanamycin; chloramphenicol acetyl- transferase (CAT) that acetylate chloramphenicol;

(3) efflux pumps that actively export antibiotics from cytoplasm out of the cell, such as the tetracycline efflux pump that is expressed in the presence of tetracycline, plus other pumps, conferring multidrug resistance, that are capable of exporting a range of antibiotics; and

(4) mutations that change the protein target of the antibiotic such that it is no longer inactivated by it; for example, beta-lactams are bactericidal because they inhibit penicillin- binding proteins (PBPs) that are required for peptidoglycan biosynthesis and bacterial cell wall integrity and PBP mutants with reduced binding to beta-lactams will not be inhibited.

Several approaches are currently being used or developed to address the problem of antibiotic resistance, including the following.

Firstly, new antibiotics, such as derivatives of existing drugs, have been developed. Fewer new antibiotic drugs have been developed, and many are more toxic so are used in the last resort. Microorganisms have acquired resistance to new antibiotics of both types.

Secondly, direct inhibition of resistance enzymes has been attempted. Examples include clavulanic acid, a beta- lactamase inhibitor which is used in combination with amoxycillin, a beta-lactam antibiotic (also called Augmentin). Other beta-lactamase antibiotic inhibitors include the carbapenems. But even here resistance has appeared. Blueberry Therapeutics and Avacta are developing peptide affimers to target mechanisms of resistance.

Development of new inhibitors of antibiotic resistance enzymes requires a long pipeline of drug development. An alternative approach has been adopted by Dr Eric Brown at McMaster University, Canada, who is screening known drugs (already approved for use) with unrelated targets, for cryptic activity in inactivating antibody resistance mechanisms.

Thirdly, non-antibiotic bactericides have been used. For example, infection by bacteriophage was developed in the 1920's and although largely discontinued with the discovery of antibiotics, has been retained in certain countries. Current approaches use virulent, lytic bacteriophage that kill bacteria, including antibiotic resistant bacteria, but this opens the way for selection of bacterial variants that are resistant to bacteriophage infection. To obviate this, preparations containing a mixture of different strains of bacteriophage are being used.

Another disadvantage of the use of such lytic bacteriophage in patients suffering from sepsis is that cell lysis and death by lytic bacteriophage can release endotoxins from the cell into the blood and can cause endotoxin shock.

Further compositions and methods for combating antibiotic resistant microorganisms are required.

According to a first aspect of the present invention, there is provided a recombinant polynucleotide comprising a clustered regularly interspaced short palindromic repeat (CRISPR) array nucleic acid sequence having or transcribing an RNA guide molecule with a spacer sequence sufficiently complementary to a target sequence of an antibiotic resistance gene in a microorganism for the antibiotic resistance gene to be inactivated in the presence of a CRISPR associated (Cas) DNA-binding polypeptide or a functional equivalent or a modified version thereof, thereby sensitising the microorganism to the antibiotic. In another aspect of the invention, there is provided a delivery vehicle for introducing a polynucleotide into a microorganism, such as an antibiotic-resistant microorganism, in which the delivery vehicle comprises a recombinant polynucleotide for inactivation of DNA carrying a gene encoding an antibiotic resistance enzyme which confers antibiotic resistance to the microrganism, and in which the recombinant polynucleotide comprises a clustered regularly interspaced short palindromic repeat (CRISPR) array nucleic acid sequence having or transcribing an RNA guide molecule with a spacer sequence sufficiently complementary to a target DNA sequence of the antibiotic resistance gene for the antibiotic resistance gene to be targeted and inactivated in the presence of a CRISPR associated (Cas) DNA-binding polypeptide or a functional equivalent or a modified version thereof, thereby sensitising the microorganism to the antibiotic, wherein the delivery vehicle is a non-virulent or a lysogenic bacteriophage.

One general aim of the present invention is inactivation of DNA carrying a gene encoding an antibiotic resistance enzyme using a CRISPR/Cas system. An advantage of the invention is that one or more existing antibiotics can be used to treat infectious disease, as

microorganisms become re-sensitised to the antibiotics or are prevented from acquiring antibiotic resistance.

The target sequence of an antibiotic resistance gene may be a sequence flanking the gene itself which, if disrupted, inactivates the antibiotic resistance gene. For example, if the antibiotic resistance gene is located on a plasmid, the invention may encompass a target sequence in the plasmid. In contrast to prior art approaches of inactivating antibiotic resistance enzymes, the present invention will not require new drug development and the concomitant regulatory approval required for each new drug. Rather, the invention provides a tool which can be applied to target and inactivate relevant antibiotic resistance genes directly rather than the gene products. For example, a gene encoding an antibiotic resistance enzyme, or a gene encoding a protein regulating the uptake and export of an antibiotic by altering the membrane permeability and efflux pump expression, respectively, can be targeted. The CRISPR/Cas system is an RNA-mediated genome defense pathway that is part of a natural bacterial and archaeal immune system against nucleic acid invaders, analogous to the eukaryotic RNAi pathway (see for example Grissa et al., 2007, BMC Informatics 8: 172; Horvath & Barrangou, 2010, Science, 327: 167-170; Gasiunas et al., 2012, Proc. Natl Acad. Sci. USA 109: E2579; Marraffini & Sontheimer, 2008, Science, 322: 1843-1845; Garneau et.al.,2010, Nature 468: 67). Natural CRISPR systems contain a combination of Cas genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR- mediated nucleic acid cleavage. Three types (l-lll) of CRISPR systems have been identified thus far in a wide range of bacterial and archaeal hosts. Each CRISPR locus is composed of a series of short DNA direct repeats separated by non-repetitive spacer sequences. The spacer sequences, in nature, typically originate from foreign genetic elements such as bacteriophage and plasmids. As used herein, the series of repeats plus non-repetitive spacer sequences is known as a CRISPR array. The CRISPR array is transcribed and hybridised with repeat complementary tracrRNA followed by cleavage within the direct repeats and processed into short mature dual tracrRNA:crRNAs containing individual spacer sequences, which direct Cas nucleases to a target site (also known as a "protospacer"). For example, the Type II CRISPR/Cas9 system, a well-studied example, carries out a targeted DNA double- strand break ("DSB") in four steps. Firstly, two RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Secondly, tracrRNA hybridises to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs (also referred to herein as "RNA guide molecules gRNA" containing individual or monomer spacer sequences. Thirdly, the mature crRNA:tracrRNA complex directs Cas9 protein in the form of a ribonucleoprotein to the target DNA via base-pairing between the spacer on the crRNA and the target site on the target DNA. Finally, Cas9 mediates cleavage of target DNA and creates a DSB.

In the present invention, as elaborated herein, modified CRISPR constructs are used to target antibiotic resistance genes. The recombinant polynucleotide of the invention using such a construct is also referred to herein as an "assassin construct" which is used to effect inactivation of such genes.

The main focus of using CRISPR technology to date has been for use as a DNA editing tool for reverse genetics, primarily in eukaryotes. However, WO2007/025097 describes the use of CRISPR technology for modulating resistance in a cell against an invading target nucleic acid or a transcription product thereof, especially against invading bacteriophages. Methods for downregulating prokaryotic gene expression using CRISPR technology to target mRNA transcribed by the genes have been suggested for example in WO2010/075424.

WO2012/164565 describes a CRISPR system from Lactoccocus and use of the system for modulating resistance of a cell against an invading target nucleic acid or a transcription product thereof. The present invention, by contrast, concerns inter alia inactivation in an antibiotic-resistant microorganism of genes involved in conferring the antibiotic resistance. According to the invention, the RNA guide molecule may mediate binding of the Cas DNA- binding polypeptide or its functional equivalent or its modified version to the antibiotic resistance gene. This mirrors the natural system described above.

The Cas DNA-binding polypeptide or its functional equivalent or its modified version of the invention may also be capable of binding to RNA or other nucleic acid molecules. In other words, the requirement for the Cas DNA-binding polypeptide or its functional equivalent or its modified version to be capable of binding DNA does in some aspects of the invention does not exclude the polypepeptide or its functional equivalent or its modified version being capable of binding RNA or other nucleic acid molecules. In these aspects, the Cas DNA- binding polypeptide or its functional equivalent or its modified version may be referred to as a Cas nucleic acid-binding polypeptide or its functional equivalent or its modified version.

For certain applications, the microorganism may have a natural endogenous, or introduced engineered, Cas DNA-binding polypeptide or functional equivalent or modified version. This means that the recombinant polynucleotide of the invention is not required to encode the Cas DNA-binding polypeptide or functional equivalent or modified version. Alternatively, the recombinant polynucleotide of the invention may further comprise a nucleic acid sequence which encodes the Cas DNA-binding polypeptide or its functional equivalent or modified version. In another aspect, the recombinant polynucleotide of the invention does not encode the Cas DNA-binding polypeptide or its functional equivalent or modified version but may be used in conjunction with a separate polynucleotide which does. Other means for introducing the Cas DNA-binding polypeptide or its functional equivalent or its modified version into the microorganism may be used.

An exemplar Cas DNA-binding polypeptide according to the invention is Cas9 or a functional equivalent thereof or a modified version thereof.

In the recombinant polynucleotide according to various aspects of the invention, the CRISPR array nucleic acid sequence may have or transcribe additional RNA guide molecules each comprising a spacer sequence sufficiently complementary to a target sequence of the antibiotic resistance gene or one or more additional antibiotic resistance genes. The or each RNA guide molecule may be transcribed from its own promoter sequence. Alternatively, a set of a number of RNA guide molecules may be transcribed from one promoter sequence and optionally in combination with one or more other such sets. For example, a set of four RNA guide molecules may be transcribed from one promoter sequence, for example in combination with one or more other such sets of guide molecules.

Having multiple RNA guide molecules allows different antibiotic resistance (or other types of) genes in a microorganism to be targeted and inactivated simultaneously. The recombinant polynucleotide according to various aspects of the invention may additionally or alternatively be designed to include an RNA guide molecule (such as a further RNA guide molecule) targeting a gene involved in pathogenicity or other aspects of microbial metabolism. For example, certain pathogens form biofilms that make it difficult for antibiotics to gain access to them. One or more genes involved in bacterial metabolism for biofilm production may be targeted.

Spacer sequence distal from a promoter are typically less efficiently transcribed. Ideally, multiple RNA guide molecules to different targets should be more or less equally

represented. Thus, one promoter transcribing each RNA guide molecule may be used (instead of relying on a long polycistronic RNA guide molecule [or precursor crRNA] transcription).

For example, there are many resistance genes encoding beta-lactamases (bla genes) giving resistance to a large range of different beta-lactam antibiotics. DNA constructs expressing multiple RNA guide molecules, which may each be individually transcribed from their own such promoters, may be used to target a number of different bla genes. Thus in aspects of the invention, the CRISPR array nucleic acid sequence may have or transcribe one or more RNA guide molecules each comprising a spacer sequence sufficiently complementary to a target sequence of one or more beta-lactamase genes. For example, the one or more RNA guide molecules may target one or more or all of the genes selected from the group consisting of : NDM, VIM, IMP, KPC, OXA, TEM, SHV, CTX, OKP, LEN, GES, MIR, ACT, ACC, CMY, LAT, and FOX.

In particular, the one or more RNA guide molecules may comprise a spacer sequence sufficiently complementary to target sequences of the beta lactam family of antibiotic resistance genes, including one or more or all of the following: a first spacer sequence sufficiently complementary to target sequences for NDM-1 , -2, -10; a second spacer sufficiently complementary to target sequences for VIM-1 , -2, -4, -12, -19, -26, -27- 33, 34; a third spacer sufficiently complementary to target sequences for IMP-32, -38, -48; a fourth spacer sufficiently complementary to target sequences for KPC- 1 , -2 , -3 , -4 , -6 , -7 , -8 , - 1 1 , -12 , -14 , -15 , -16, -17; a fifth spacer sufficiently complementary to target sequences for OXA-48; a sixth spacer sufficiently complementary to target sequences for TEM-1 ,-1 B, -3, - 139, -162, -183, -192, -197, -198, -209, a seventh spacer sufficiently complementary to target sequences for SHV and its variants; and an eighth spacer sufficiently complementary to target sequences for CTX and its variants.

The antibiotic resistance gene to be inactivated may be located on a chromosome, or on an extrachromosomal replicating DNA molecule known as a replicon and including plasmids and bacteriophage.

The CRISPR/Cas system used according to an aspect of the invention generates a DSB in the target sequence. Where the target sequence is located on a chromosome or a replicon such as a bacterial chromosome or plasmid, then a DSB can lead to degradation and hence loss of the chromosome or replicon suffering such a DSB. If the target sequence is located on a bacterial chromosome then the cell may die directly as a consequence of the DSB. Additionally, some plasmids (including natural plasmids) carry killing functions that only become toxic if the cell loses the plasmid, which is a natural mechanism to ensure faithful inheritance of plasmids in dividing cells. If a plasmid carrying the target sequence of the antibiotic resistance gene also carries such a killing function, and the plasmid is lost as a result of the DSB generated, the cell may die (see Sengupta & Austin, 201 1 , Infect. Immun. 79: 2502-2509).

In the event that cell death caused by such DSB increases selection pressure for resistance against the recombinant polynucleotide according to various aspects of the present invention, this may be mitigated by, for example, employing a modified Cas DNA-binding polypeptide which seals the target site after generating a deletion to inactivate the target sequence of the antibiotic resistance gene, rather than generate a DSB.

Thus the Cas DNA-binding polypeptide according to various aspects of the invention may in certain aspects be substituted by a modified Cas DNA-binding polypeptide comprising a recombinase catalytic domain, wherein the modified Cas DNA-binding polypeptide does not generate DSBs but creates a deletion and reseals a site in the target sequence.

The modified Cas DNA-binding polypeptide may for example be a modified Cas9 protein comprising a recombinase catalytic domain.

The recombinant polynucleotide according to various aspects of the invention may further comprise a nucleotide sequence which encodes a gene conferring a selective advantage to the microorganism, for example thereby increasing the efficiency of delivery of the

CRISPR/Cas system to the target microorganism. For example, the gene may confer a growth advantage over non-infected siblings, or genes encoding a bacteriocin - these are protein toxins produced by bacteria to kill or inhibit growth of other bacteria - and corresponding immunity polypeptide may be used. The selective advantage to the microorganism may include or be one which prevents or diminishes the effect of loss of a replicon due to a DSB caused by Cas DNA-binding polypeptide. For example, the nucleotide sequence which encodes a gene conferring a selective advantage to the microorganism may encode an antitoxin that neutralises the effect of a toxin or killer function carried by a replicon on which the target sequence is located. Also, the nucleotide sequence which encodes a gene conferring a selective advantage to the microorganism may encode one or more proteins that are encoded by a replicon subject to degradation due to a DSB caused by Cas DNA-binding polypeptide.

In another aspect of the invention, there is provided a delivery vehicle for introducing a polynucleotide into a microorganism, in which the delivery vehicle comprises the recombinant polynucleotide as defined herein. The delivery vehicle according to various some aspects of the invention may be a bacteriophage. Alternatively, the delivery vehicle may be a plasmid such as a conjugative plasmid or other plasmid replicon, a nucleotide vector, linear double-stranded DNA (dsDNA), an RNA phage or an RNA molecule.

The delivery vehicle may be a non-virulent bacteriophage, such as bacteriophage M13, a filamentous phage that infects Escherichia coli cells, replicates and is secreted from the bacterial host cell without killing the bacterial host, which continues to grow and divide more slowly. Another suitable filamentous phage is NgoPhi6 isolated from Neisseria gonorrhoeae that is capable of infecting a variety of Gram negative bacteria without killing the host.

Alternatively, lysogenic phage may be used that do not always kills host cells following infection, but instead infect and become dormant prophage. This invention may thus use a novel system to inactivate antibiotic resistance genes in bacteria primarily using bacteriophage that do not kill bacteria, and/or conjugative plasmids and/or direct DNA transformation, as the delivery mechanisms for the recombinant polynucleotide of the invention. In a further aspect of the invention, there is provided a composition comprising the recombinant polynucleotide as defined herein, or the delivery vehicle as defined herein.

The composition may be a pharmaceutical composition, a non-pathogenic microorganism such as a commensal bacterium for example in a probiotic formulation, or a dietary supplement.

As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active agents (such the recombinant polynucleotide or the delivery vehicle as described herein) with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active agent to an organism.

The composition may be formulated for topical, enteral or parenteral administration. Compositions of the present invention may, if desired, be presented in a pack, dispenser device or kit, each of which may contain one or more unit dosage forms containing the active agent(s). The pack, dispenser device or kit may be accompanied by instructions for administration.

Also provided according to the invention is the composition as defined herein for use as a medicament.

In another aspect there is provided according to the invention a composition as defined herein for use in the treatment or prevention of an infection caused by an antibiotic-resistant microorganism comprising an antibiotic resistance gene targeted by the RNA guide molecule of the recombinant polynucleotide.

The invention further provides a method of treating or preventing an infection in a subject caused by an antibiotic-resistant microorganism comprising an antibiotic resistance gene, in which the method comprises the step of introducing into the microorganism a therapeutically effective amount of the composition as defined herein where the RNA guide molecule targets the antibiotic resistance gene, thereby inactivating the antibiotic resistance gene and sensitising the microorganism to the antibiotic.

The composition may be administered topically or orally. Alternatively the composition may be administered by intravenous, parenteral, ophthalmic or pulmonary administration.

Compositions of the present invention for administration topically can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion or dusting powder. Compositions provided herein may be formulated for administration by inhalation. For example, the compositions may be in a form as an aerosol, a mist or a powder. Thus compositions described herein may be delivered in the form of an aerosol spray presentation from pressurised packs or a nebuliser, with the use of a suitable propellant such as for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Where using a pressurised aerosol, a dosage unit may be determined by providing a valve to deliver a metered amount.

In the method, the subject may be a plant, a fish, a bird, a reptile or a mammal (such as a human). According to the method, the delivery vehicle may be transferred from the microorganism directly into another microorganism (such as antibiotic-resistant microorganism) by plasmid conjugation or bacteiophage infection. The method may further comprise a step of simultaneously or subsequently administering to the subject an antibiotic to which a microorganism has become sensitised.

A further aspect of the invention provides a method of inactivating antibiotic resistance in a microorganism, the method comprising introducing into the microorganism of the recombinant polynucleotide as defined herein, or the delivery vehicle as defined herein. The method may be an in vivo method applied to a subject, or an in vitro method.

Also provided according to the invention is a host cell comprising the recombinant polynucleotide defined here. The host cell may, for example, be a commensal bacterium.

Microorganisms targeted by various aspects of the invention may be on a body surface, localised (for example, contained within an organ, at a site of a surgical wound or other wound, within an abscess), or may be systemic. Included is the treatment of bacterial infections that are amenable to therapy by topical application for example using

bacteriophage of the invention.

The present invention also encompasses coating of surfaces other than body surfaces with the recombinant polynucleotide, delivery vehicle or composition of the present invention, for example wound dressings or medical device surfaces.

Further aspects, features and non-limiting embodiments of the present invention will now be described below with reference to the following drawings:

Figure 1. CRISPR/Cas9-mediated bacterial immunisation against antibiotic resistant genes on a plasmid: I, CRISPR/Cas locus, where boxes denote different spacer sequences targeting different antibiotic resistance genes; II, gRNA-Cas9 where boxes denote different gRNAs targeting each antibiotic resistance gene; III, plasmid harbouring antibiotic resistance gene; IV, target recognition and positioning of Cas9; V, cleaved plasmid, VI: bacteriophage. Ap ^R = ampicillin resistant, Cm ^R = chloramphenicol resistant, Km ^R = kanamycin resistant. Ap ^s = ampicillin sensitive, Cm ^s = chloramphenicol sensitive, Km ^s = kanamycin sensitive. Bla= beta-lactamase, CAT = chloramphenicol acetyl transferase, APH = aminoglycoside phosphotransferase. 1. Injection - CRISPR/Cas9 is injected into the bacterial cell along with phage DNA by bacteria-specific phage infection. 2. Lysogenisation - Phage DNA is lysogenised and integrates into the bacterial host chromosome (B. chr.). 3. crRNA biogenesis and assembly of Cas9 - Pre-crRNA is transcribed and hybridised with tracrRNA and processed to make mature crRNA:tracrRNA (an RNA guide molecule, or "gRNA"), which is assembled with Cas9. 4. Recognition - These gRNA-Cas9 complexes recognise target DNA on the plasmid. 5. Cleavage - The gRNA-Cas9 complexes cleave DNA at the sites recognised by crRNAs. 6. Inactivation - This leads to inactivation of the production of antibiotic resistant enzymes; 7. Sensitive - Thus, the bacterial cell becomes sensitive to various antibiotics.

Figure 2. Preventing non-pathogenic bacteria and asymptomatic pathogens from a future encounter with antibiotic resistance genes: I, CRISPR/Cas locus, exemplified here as present in the bacterial chromosome (B. Chr.) and where boxes denote different spacer sequences targeting different antibiotic resistance genes; II, gRNA-Cas9, where boxes denote different gRNAs targeting each antibiotic resistance gene; III, relaxase -an enzyme that makes a strand-specific, sequence-specific nick in double-stranded DNA to initiate conjugal transfer of plasmid DNA; IV, bacteriophage, V, plasmid; VI, double-stranded DNA; VII, single-stranded DNA. Three entry pathways to encounter future antibiotic resistance genes are shown. 1. Transformation- Entry of naked DNA carrying antibiotic resistance gene into the bacterial cell, 2. Bacteriophage-mediated transduction- Phage carrying antibiotic resistance gene infects a bacterial cell. 3 Conjugation- Plasmid carrying antibiotic resistance gene is conjugally transferred from a donor bacterial cell to a recipient bacterial cell. When these antibiotic resistance genes are introduced into the "immunised" cell; it is pre-armed with a CRISPR/Cas locus encoding a gRNA-Cas9 that finds the specific target sequence on the antibiotic resistance gene and cleaves it to disrupt the gene.

Figure 3. Re-sensitising symptomatic pathogens to antibiotics: I, CRISPR/Cas locus in the bacterial chromosome (B. Chr.), where boxes denote different spacer sequences targeting each antibiotic resistance gene; II, gRNA-Cas9, where boxes denote different gRNAs targeting each antibiotic resistance gene; III, antibiotics; IV, protein synthesis is disrupted by cleaving corresponding gene; V, plasmid. This figure shows an asymptomatic pathogen becoming symptomatic because of an opportunistic infection. The pathogen contains plasmid DNA that provides various antibiotic resistance agents such as 1. Efflux pumps- capable of pumping antibiotics out of the bacterial cell, 2 Antibiotic degradation enzymes-and 3. Antibiotic modification enzymes. Each antibiotic resistant agent is disrupted (X) by gRNA-Cas9-mediated site-specific cleavage of corresponding genes.

Figure 4. Three possible CRISPR/Cas delivery routes: I, CRISPR/Cas locus where boxes denote different spacer sequences targeting each antibiotic resistance gene; II, relaxase -an enzyme that makes a strand-specific, sequence-specific nick in double-stranded DNA to initiate conjugal transfer of plasmid DNA; III, bacteriophage; IV, plasmid; V, double-stranded DNA; VI, single-stranded DNA. This figure shows three possible delivery routes of

CRISPR/Cas9 expression construct. 1. Transformation- Entry of naked DNA carrying CRISPR/Cas9 into the bacterial cell via a DNA receptor on the cell surface, followed by integration (4) into the bacterial chromosome (B. Chr.) , 2. Bacteriophage-mediated transduction- Phage carrying CRISPR/Cas9 infects a bacterial cell, followed by

circularisation (5) and then phage-mediated integration (4) into the bacterial chromosome (B. Chr.). 3 Conjugation- Plasmid carrying CRISPR/Cas9 is conjugally transferred from a donor bacterial cell to a recipient bacterial cell, followed by circularisation (5) and plasmid replication. In all cases, boxes in the CRISPR/Cas9 locus denote different spacer sequences targeting each antibiotic resistance gene.

Figure 5. Structure of bidirectional antibiotic resistant transmission model (see also Am J Epidemiol. 2013;178(4):508-520) and the effect of antibiotic resistance gene inactivation therapy: A, Antibiotic Resistance is Dominant; B, Antibiotic Sensitivity is Dominant; S, Susceptible, AS, Antibiotic Sensitive; AR, Antibiotic Resistant; T,

Transmission; R, Recovery, C, Conversion, CCD, CRISPR/Cas9 delivery. Antibiotic resistance genes in an antibiotic resistant state and the encounter of antibiotic resistance genes in a sensitive state are disrupted for example by gRNA-mediated Cas9 cleavage. Antibiotic resistance gene inactivation therapy converts the bidirectional model (A) to an almost unidirectional conversion model (B) by increasing the conversion rate from antibiotic resistant to sensitive and decreasing the conversion rate from antibiotic sensitive to resistant. The area of each square represents the population density of each state. The width of the arrows is proportional to the magnitude of each parameter (transmission, recovery and conversion), i.e. transmission of antibiotic resistant bacteria is assumed to be higher than antibiotic sensitive bacteria in this figure. Recovery from the infection of pathogens sensitive to antibiotics is higher than for antibiotic resistant pathogens. In the presence of antibiotics, antibiotic-sensitive pathogens are converted to antibiotic resistant pathogens much more than the reverse conversion from resistant to sensitive pathogens because of the selection pressure. The treatment aim is to reverse these conversion parameters to drive the antibiotic resistant pathogen population to a sensitive state by introducing CRISPR/Cas9 into the bacteria.

Figure 6. Structure of superinfection antibiotic resistant transmission model and the effect of antibiotic resistance gene inactivation therapy: The bidirectional antibiotic resistance transmission model depicted in Figure 5 can additionally or alternatively be described by the following superinfection antibiotic resistance transmission model (see also Am J Epidemiol. 2013;178(4):508-520, as for Figure 5). When the antibiotic resistance genes are disrupted for example by gRNA-mediated Cas9 cleavage, this allows the population shift from the antibiotic resistant state to the antibiotic sensitive state via the superinfection state. Disruption of antibiotic resistance genes increases the conversion rate from the antibiotic resistant to the sensitive state. This figure gives a comparison of the relative population density before (Figure 6A) and after (Figure 6B) applying the CRISPR-Cas system. The population is composed of S, Susceptible; Iw, sensitive; Iz, resistant; and Iwz, superinfection state. The area of each circle is proportional to the relative population density in each state. In this simulation, initial density in each state is assumed to be identical (0.25 each, represented by a thin-lined circle). Thick lined circles represent the population density at the equilibrium state. CRISPR-Cas system is clearly contributing to an increase of the population in the susceptible state, thus the recovery rate in the equilibrium state (i.e. S is expanding in B) and to reduce the population in the resistant state (i.e. Iz is shrinking in B). The width of the arrows is proportional to the magnitude of each parameter (infection, recovery and state conversion), i.e. the infection of antibiotic resistant bacteria is assumed to be lower than that of antibiotic sensitive bacteria in this figure. Recovery from the infection of the antibiotic sensitive pathogens is higher than that from the infection of the antibiotic resistant pathogens, because antibiotics are effective on the sensitive strains. In the presence of antibiotics, the antibiotic sensitive pathogens are converted to the antibiotic resistant pathogens much more than the reverse conversion because of the antibiotic selection pressure. The aim of the treatment is to reverse these conversion parameters and to drive the antibiotic resistant pathogen population to the sensitive state.

Figure 7. Structure of CRISPR locus: This figure shows CRISPR locus containing six spacers targeting six different regions. Each crRNA is transcribed monocistronically from the same promoters denoted P to control the transcription level identical for each target. Each crRNA transcript starts with a leader sequence L and terminates with a terminator sequence T. Transcripts of each pre-crRNA are shown as arrows and boxes containing different spacer sequences are indicated by unique shading.

Figure 8. Mapping short bacterial off-target sequence on the bla gene sequence. The figure shows the local alignment of bacterial short sequences (SEQ ID NOs: 3-5, 8-14, 17-23 and 26-33) mapped to the beta lactamase gene. Beta lactamase sequence (SEQ ID NOs: 1 - 2, 6-7, 15-16 and 24-25) is shown in grey in the top of each panel, PAM (protospacer adjacent motif) sequence is shown in black. Base pairing region with crRNA is underlined, off-target seed sequence on the bacterial genome is italicised. Off-target seed sequence, including PAM sequence, is indicated by two vertical lines. Two numbers on the sequence of beta lactamase gene are the expected two base positions where the phosphodiester bond is cleaved by Cas9 (see Example 1 below).

Figure 9. Predicted crRNA secondary structure. With reference to Figure 8 and Example 1 , predicted secondary structures of the crRNA sequences using mFold are shown. The sequences of CR05, CR30, CR70 and CR90 shown in Figure 9 correspond to SEQ ID NOs: 34-37, respectively.

Figure 10. Expected cleavage positions on beta-lactamase gene from pBR322 (SEQ ID NO: 38). Two protospacer sequences, which are base-pairing with crRNA spacer sequences, are underlined (protospacer strand). Two anti-protospacer sequences, which are displaced when protospacer sequences are base pairing with crRNA spacer sequences, are bold italicised (anti-protospacer strand). Associated PAM sequences are indicated either boxed small letters (on protospacer strand) or boxed capital letters (on anti-protospacer strand). Expected cleavage positions are indicated by asterisks. Leader sequence is underlined from 1 st to 69th base and highlighted in grey.

Figure 11. Photograph of electrophoretically separated DNA products on a 0.8% agarose gel showing PCR amplicons generated in Example 2 from each of the three regions plus the EcoRV digested vector pACYC184. The marked lanes are as follows: 1 ^* - NEB (N3232S) 1 Kb molecular markers, 2 μg/lane; 2 - Fragment 1 , tracrRNA-Cas9 region : 4758 bp; 3 ^* - NEB (N3232S) 1 Kb molecular markers, 0.75 μg/lane; 4 - pACYCI 84 uncut; 5 - pACYCI 84 EcoRV cut : 4245 bp; 6 - pACYCI 84 EcoRV cut : 4245 bp; T - NEB (N3232S) 1 Kb molecular markers, 0.12 μg/lane; 8 - Fragment 2, leader and first direct repeat region : 276 bp; 9 ^* - NEB (N3232S) 1 Kb molecular markers, 0.2 μg/lane; and 10 - Fragment 3, Second direct repeat region : 452 bp. Note: ^* The mass of the each PCR amplicon is estimated by comparing the intensity of the appropriate marker band using NIH ImageJ 1 .48v software(http://imagej. nih.gov/ij/).

Figure 12. Plasmid map of pNB100 constructed in Example 2. The plasmid map was drawn by SnapGene viewer ver. 2.4.3 free version (http://www.snapgene.com). Two direct repeats (DR) are shown as narrow white rectanglular boxes adjacent to the 3' end of leader sequence.

Figure 13. Photographs show results of "Nemesis symbiotic activity" (NSA) according to an embodiment of the invention by bacterial cell mating (see Example 2). The left plate shows JA200 pNT3 x DH5a pNB100 in Ap100Cm35, while the right plate shows JA200 pNT3 x DH5a pNB102 in Ap100Cm35, both plated at 5 x 10 ⁷ cells /ml.

Figure 14. Photographs show results of NSA according to another embodiment of the invention by plasmid transformation (see Example 2). Left: LB Cm35 plate. Colonies 1 - 40 are DH5a pBR322 transformed with pNB102; Colonies 45-60 are DH5a pBR322 transformed with pNB100. All colonies show resistance to Cm carried on plasmids pNB100 and pNB102; Right: LB Ap100 plate. Note that colonies 1 -40 have lost ApR following transformation with pNB102 carrying the spacer region targeted against the beta-lactamase gene carried on the plasmid pBR322 in strain NBEcOOI , thereby demonstrating Nemesis symbiotic activity. pNB100 lacking this spacer region but carrying the Cas9 gene is unable to inactivate the beta-lactamase gene.

Figure 15. Shows a phagemid of Ngophi6. ORF1 1 and ORF7 of Ngophi6 are deleted from Ngophi6 genome. Coding sequences are represented by the arrows indicating the translation polarity of each ORF. The corresponding gene nomencrature of each Ngophi6 phage ORF to M13 are ORF1 (g II), ORF2 (gV), ORF4 (gVIII), ORFV (gVIII), ORF8 (gVI), ORF9 (gl). M13 gene nomencretures are in the parenthesis. The second T of the putative left integration site L = CTTATAT is changed to A to give L' = CATATAT to avoid integration. MCS = Multiple cloning site. The location of Ngophi6 and M13mp18 are indicated by the large two open arrows.

Figure 16. Shows a set of spacer sequences (SEQ ID NOs: 39-60) that encode 20 guide RNA molecules targeted against 1 17 different bla genes identified in the NCBI ARDB database for Klebsiella pneumoniae (see Example 5). Candidate spacer sequences were identified to disrupt all the Klebsiella pneumoniae beta lactamase genes found in the ARDB database. Beta lactamase gene sequences are collected from the ARDB database with the keyword Klebsiella pneumoniae. Redundant sequences were removed and unique sequences used for multiple sequence alignment using web program Clustal Omega. One canonical sequence was chosen from each cluster and the 20 nt spacer sequences predicted by the web program Jack Lin's CRISPR/Cas9 gRNA finder were collected. The spacer sequence is chosen to maximise the ratio of the proto-spacer sequence found in the sequences belonging to the same branch. Thus each of the example spacer sequences shown in the 4 ^th column has the capability to disrupt the genes in the third column. Beta lactamase genes used in this analysis are: SHV-a = 1 , 2, 2a, 5, 5a, 1 1 , 12, 14, 26, 27, 28, 31 , 33, 38, 43, 44, 48, 55, 56, 60, 61 , 62, 71 , 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82,85, 89, 92, 98, 99, 101 , 103, 106, 107, 108, 109, CTXM-b = 1 , 3, 10, 12, 15, 22, 32, 54, 60, 62, 68, CTXM-c = 13, 14, 16, 18, 19, 24, 26, CTXM-d = 2, 35, 59, CTXM-e = 26, 63, TEM-f = 1 , 1 b, 3, ESBL, 139, KPC-g = 1 , 2, 3, 4, OKP-h = A1 1 , A12, A16, A17, B13, B-SB613, 6, LEN-i = 2, 17, 18, 19, 20, 21 , 22, 24,GES-j = 1 , 3, 4, 5, VIM-a = 1 , 2, 4, 12, 19, IMP-b = 4, 8, CMY-a = 2, 4, 25, 31 , 36, LAT-b = 1 , 2, CMY-c = 1 , 8b, 10, 19, FOX-d = 1 , 5, 7, OXA-a = 1 , 30, 47, OXA-2, OXA-9, OXA-b = 10, 17. Beta lactam antibiotics are classified into four classes, penams, cephems, carbapenems and monobactams. One antibiotic name is listed as an example under each class. The beta lactamase, which can open the beta lactam ring is indicated by R. For example, carbapenem is inactivated by KPC. If it is desired to re- sensitise bacteria to carbapenem, the spacer sequence 5'-TTGTTGCTGAAGGAGTTGGG should be employed into spacer array to inactivate KPC genes. Note that the spacer sequence for CMY-a can also be employed for LAT-b cleavage. The example of spacer sequences are shown from 5' to 3' direction.

Figure 17. Shows a set of spacer sequences (SEQ ID NOs: 61 -77) that encode 17 guide RNA molecules targeted against 154 different bla genes identified in the CARD database for Klebsiella pneumoniae (see Example 5). Candidate spacer sequences were identified to disrupt all the Klebsiella pneumoniae beta lactamase genes found in the CARD database. This table was created with the same method explained in the figure legend in Figure 16. The example of spacer sequences are shown from 5' to 3' direction.

Figure 18. Map of a modified Cas DNA-binding polypeptide, Cas9R. A genetic fusion between Cas9 and Tn3 resolvase. Resolvase and Cas9 are indicated by arrows. The direction of the arrowhead represents the transcription polarity. Functional domain names of Cas9 are shown in the boxes below Cas9 open arrow. This Cas9 is the endonuclease activity deficient mutant dCas9, with amino acid substitutions D10A in RuvCI domain, H840A in HNH domain (as described by Tsai et al. [2014, Nature Biotechnology 32: 569-576]). A mutant Tn3 resolvase (as described by Proudfoot et al. [201 1 PLoS ONE 6(4): e19537]) is fused to the N-terminus of this dCas9 via a 12 mer polypeptide linker. Positions of some of these substituted amino acid residues reducing the specificity of the recombination site are indicated by short vertical bars in the N terminal domain, residues 1 -148, of the resolvase. The full list of these substitutions is: R2A, E56K, G101 S, D102Y, M103I, Q105L, V107F, E132A, L135R. In the Cas9 regions of the fusion: RuvCI, II, III, HNH and PI (PAM interaction) domains are nuclease domains, REC1 a and REC1 b are recognition domains of repeat and anti-repeat RNA, respectively. REC2 domain does not have any contact to the protospacer-gRNA heteroduplex. Four CRISPR spacer sequences S1 , S2, S3 and S4 are arrayed under the expression of one CRISPR leader sequence and are required to bring about the Cas9R-mediated recombination event by the mutant Tn3 resolvase leading to the deletion and re-ligation of the target sequence. Tn3R = Tn3 Resolvase, R = Direct repeat, L = Leader sequence. Figure 19. Schematic showing site-specific positioning of resolvase by gRNA directed Cas9. The open arrow in step I is the target antibiotic resistance gene on the plasmid for inactivation. Each recombination site A (A1 , A2) and B (B1 , B2) are recognised by gRNA independently and correctly positioned resolvases are dimerised in close proximity (step II). 5 Dimers in each recombination site A1 +A2 and B1 +B2 are tetramerised to form a synapse (step III). The synaptic complex (III) is enlarged, gRNAs are presented as dotted arrows designated S1 , S2, S3 and S4. Large ovals represent dCas9, longitudinal ovals are resolvases connected via linker peptides. White and grey longitudinal ovals are resolvase catalytic domains dimerising on the recombination site B and A, respectively. The vertical 10 arrows indicate the cleavage position on the recombination sites by resolvase. The thin

horizontal parallel arrows represent DNA containing the recombination site A1 +A2 and the thick horizontal parallel arrows represent DNA containing the recombination sites B1 +B2. The arrowhead shows the 3' end of the DNA sequence. Short black block arrows are locations of each of the PAM sequences.

15

Figure 20. Schematic showing exchanging half site of the recombination site A1+A2 and B1 +B2 followed by strand resolution and sealing break point. Half-site of recombination A1 and B1 are exchanged and ligated and resolved. The region of the target 20 antibiotic gene is resolved as a circular DNA, while the rest of the chromosomal or plasmid replicon is re-circularised (step IV). Short black block arrows are locations of each PAM sequences after resolution.

25 Figure 21. Shows a set of spacer sequences (SEQ ID NOs: 78-85) that encode 8 guide RNA molecules targeted against the class A genes, SHV-a, CTX-M-b, TEM-c, KPC-d; the class B genes VIM-e, IMP-f , NDM-g and the class D gene, OXA-48 where SHV-a = 1 , 1 a, 2, 2a, 5, 5a, 1 1 , 12, 14, 18, 20, 21 , 22, 23, 26, 27, 28, 31 , 32, 33, 38, 43, 44, 48, 52, 55, 56, 60, 61 , 62, 71 , 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 85, 89, 92, 98, 99, 100, 101 , 103, 106, 107,

30 108, 109, 1 10, 1 1 1 , 121 , 136, 134, 137, 140, 143, 144, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 168, 172, 173, 178, 179; CTXM-b = 1 , 3, 10, 12, 15, 19, 22, 32, 52, 54, 59, 60, 62, 68, 71 , 80, 81 , 99, 141 , 147; TEM-c = 1 , 1 B, 3, 139, 162, 183, 192, 197, 198, 209; KPC-d = 1 , 2, 3, 4, 6, 7, 8, 1 1 , 12, 14, 15, 16, 17 ; VIM-e = 1 , 2, 4, 19, 26, 27, 33, 34; IMP-f = 4, 8, 32, 38; and NDM-g = 1 , 9, 10 (see Example 7). Figure 22. Shows a table giving the sequences (SEQ ID NOs: 86-98) of the oligonucleotides used in the construction of plasmids pNB200, 202, 203, 104A, 104B and 108 (see Example 7).

Figure 23. Plasmid map of pNB104A constructed in Example 7. The plasmid map was drawn by SnapGene viewer ver. 2.4.3 free version (http://www.snapgene.com). The tetramer spacer concatemer (Figure 29A) was digested with Bsa\, whose restriction site is located in A1 and A2, and ligated to Bsa\ spacer cloning sites on pNB202 to give pNB203. The single promoter and spacer region (6221 -7001 ) on pNB104A is shown. P = Promoter, L = Leader, R = Direct repeat, S = Spacer, T = Tail. The concatenated spacers (targeted against NDM, IMP, VIM and KPC) are located downstream of the single promoter.

Figure 24. Plasmid map of pNB104B constructed in Example 7. The plasmid map was drawn by SnapGene viewer ver. 2.4.3 free version (http://www.snapgene.com). The single promoter regions (6221 -6987) on pNB104B is shown. P = Promoter, L = Leader, R = Direct repeat, S = Spacer, T = Tail. The concatenated spacers (targeted against OXA-48, SHV, TEM and CTX-M) are located under expression from the single promoter.

Figure 25. Plasmid map of pNB108 constructed in Example 7. The plasmid map was drawn by SnapGene viewer ver. 2.4.3 free version (http://www.snapgene.com). The octamer spacer concatemer (Figure 29B) was digested with Bsa\, whose restriction site is located in A1 and A2, and ligated to Bsa\ spacer cloning sites on pNB100 to give pNB108. The single promoter and spacer region (6221 -7225) on pNB108 is shown. P = Promoter, L = Leader, R = Direct repeat, S = Spacer, T = Tail. The concatenated spacers (targeted against NDM, IMP, VIM, KPC, OXA-48, SHV, TEM and CTX-M) are located under the single promoter.

Figure 26. Plasmid map of pNB200 constructed in Example 7. The plasmid map was drawn by SnapGene viewer ver. 2.4.3 free version (http://www.snapgene.com). The dual promoter cassette was synthesised by PCR from the template pNB100 with primer pair NB018 and NB019, the amplicon was digested with Bbv\ and ligated to the Bsa\ site of pNB100 to give pNB200, the small Bsa\ fragment of pNB100, from position 5776-5741 (see Figure 12) is replaced in the process. The dual promoter and two spacer cloning region (6221 -7382) on pNB200 is shown. P = Promoter, L = Leader, R = Direct repeat, S = Spacer, T = Tail. The restriction enzymes Bsa\ and Sap\ are utilised to clone upstream and downstream spacer sequences, respectively.

Figure 27. Plasmid map of pNB202 constructed in Example 7. The plasmid map was drawn by SnapGene viewer ver. 2.4.3 free version (http://www.snapgene.com). The tetramer spacer concatemer (Figure 29A) was digested with Sap\, whose restriction site is located in B1 and B2, and ligated to Sap\ spacer cloning sites on pNB200 to give pNB202. The dual promoter and spacer regions (6221 -7329) on pNB202 is shown. P = Promoter, L = Leader, R = Direct repeat, S = Spacer, T = Tail. The concatenated spacers (targeted against OXA-48, SHV, TEM and CTX-M) are located downstream of the second promoter.

Figure 28. Plasmid map of pNB203 constructed in Example 7. The plasmid map was drawn by SnapGene viewer ver. 2.4.3 free version (http://www.snapgene.com). The tetra spacer concatemer a+b+c+d shown in Figure 29A was digested with Bsa\, whose restriction site is located in A1 and A2, and ligated to Bsa\ spacer cloning sites on pNB202 to give pNB203. The dual promoter and spacer regions (6221 -7501 ) on pNB203 is shown. P = Promoter, L = Leader, R = Direct repeat, S = Spacer, T = Tail. The concatenated spacers (targeted against NDM, IMP, VIM and KPC) are located downstream of the first promoter. The concatenated spacers (targeted against OXA-48, SHV, TEM and CTX-M) are located downstream of the second promoter.

Figure 29A. Tetramer spacer concatenation in Example 7. The numbers associating oligos are corresponding to the primer numbers listed in Figure 22. Oligos are pairewise annealed between 26 and 27, 28 and 34, 35 and 31 , 32 and 36 via a, c, e and g unique spacer region (I), respectively and extended in individual tubes (II). Dimer concatemer from 26 and 27 concatenate spacer a and b. Dimer concatemer from 28 and 34 concatenate spacer b, c and d. Dimer concatemer from 35 and 31 concatenate spacer e and f . Dimer concatemer from 32 and 36 concatenate spacer f, g and h (II). Concatenated dimmers a+b and b+c+d, e+f and f+g+h are further hybridised via b and f spacer region, respectively and extended to concatenate four spacers a, b, c and d or e, f, g and h (III).

The tetramer spacer concatemer e+f+g+h was digested with Sap\, whose restriction site is located in B1 and B2, and ligated to Sap\ spacer cloning sites on pNB200 to give pNB202. Tetra spacer concatemer a+b+c+d was digested with Bsa\, whose restriction site is located in A1 and A2, and ligated to Bsa\ spacer cloning sites on pNB202 to give pNB203. a = 20 mer spacer for NDM, b = 20 mer spacer for IMP, c = 20 mer spacer for VIM, d = 20 mer spacer for KPC, e = 20 mer spacer for OXA-48, f = 20 mer spacer for SHV, g = 20 mer spacer for TEM, h = 20 mer spacer for CTX-M.

Figure 29B. Octamer spacer concatenation in Example 7. The tetramer spacer concatemer a+b+c+d and e+f+g+h were amplified with primer pair NB026 and NB029, NB030 and NB033, respectively (V), and hybridise tetra concatemer via spacer d region followed by extension to yield octamer spacer a+b+c+d+e+f+g+h. This octamer was digested with Bsa\ and ligated to Bsa\, whose restriction site is located in A1 and A2, sand ligated to Bsa\ spacer cloning sites on pNB100 to give pNB108. a = 20 mer spacer for NDM, b = 20 mer spacer for IMP, c = 20 mer spacer for VIM, d = 20 mer spacer for KPC, e = 20 mer spacer for OXA-48, f = 20 mer spacer for SHV, g = 20 mer spacer for TEM, h = 20 mer spacer for CTX-M.

Figure 30. Photographs showing results of "Nemesis symbiotic activity" (NSA) according to an embodiment of the invention by bacterial cell mating (see Example 7).

Fig 30A, top left plate shows JA200 pNT3 x DH5a pNB100 in Ap100Cm35, while top right plate shows JA200 pNT3 x DH5a pNB102 in Ap100Cm35. Fig 30B shows NCTC13440 x DH5a pNB100, the top left plate and NCTC13353 x DH5a pNB100, the top right plate; and NCTC13440 x DH5a pNB104A the bottom left plate and NCTC13353 x DH5a pNB104B, the bottom right plate all in Ap100Cm35. Fig 30C shows JA200 pNT3 x DH5a pNB100 (5-), NCTC13440 x DH5a pNB100 (2-) and NCTC13353 x DH5a pNB100 (4-), top left plate; and JA200 pNT3 x DH5a pNB108 (5/8), NCTC13440 x DH5a pNB108 (2/8) and NCTC13353 x DH5a pNB108 (4/8), top right plate and JA200 pNT3 x DH5a pNB100 (5+), bottom plate, all in Ap100Cm35.

As used herein, the term "antibiotic" refers to a classical antibiotic that is produced by a microorganism that is antagonistic to the growth of other microorganisms and also encompasses more generally an antimicrobial agent that is capable of killing or inhibiting the growth of a microorganism, including chemically synthesised versions and variants of naturally occurring antibiotics.

The term "sufficiently complementary" means that the sequence identity of the spacer sequence and the target sequence is such that the RNA guide molecule comprising the spacer sequence is able to hybridise, preferably specifically and selectively, with the target sequence, thereby allowing for inactivation of the antibiotic resistance gene comprising the target sequence via the CRISPR/Cas system described herein. For example, the spacer sequence may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over its entire length with the target sequence. The term "functional equivalent" as used herein refers to a polypeptide which is capable of the same activity as a Cas DNA-binding polypeptide (or, as used herein, a Cas nucleic acid-binding polypeptide). The "functional equivalent" may have the same qualitative biological property as the Cas DNA-binding polypeptide. "Functional equivalents" include, but are not limited to, fragments or derivatives of a native Cas DNA-binding polypeptide and its fragments, provided that the equivalents have a biological activity in common with a corresponding native sequence polypeptide. Although structural identity is not necessarily required for common biological activity, in one aspect the functional equivalent may have at least 50%, 55%, 60% 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over its entire length with a Cas DNA-binding polypeptide, for example Cas9 (Ferretti et al, 2001 , PNAS, 98 No. 8: 4658-4663, Gene ID: 901 176, Cas9 Gl: 15675041 ; SEQ ID NO: 99).

The term "Cas DNA-binding polypeptide" encompasses a full-length Cas polypeptide, an enzymatically active fragment of a Cas polypeptide, and enzymatically active derivatives of a Cas polypeptide or fragment thereof. Suitable derivatives of a Cas polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of a Cas protein or a fragment thereof.

The term "modified" Cas DNA-binding polypeptide encompasses Cas DNA-binding polypeptides as defined above except that the DSB catalytic function of the polypeptide is replaced by a DNA sealing function due for example to the presence of a recombinase catalytic domain. Further features of such modified Cas DNA-binding polypeptides are described herein. Sequence identity between nucleotide or amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids or bases at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.

Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include MatGat (Campanella et al., 2003, BMC Bioinformatics 4: 29; program available from http://bitincka.com/ledion/matgat), Gap

(Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453), FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410; program available from http://www.ebi.ac.uk/fasta), Clustal W 2.0 and X 2.0 (Larkin et al., 2007, Bioinformatics 23: 2947-2948; program available from http://www.ebi.ac.uk/tools/clustalw2) and EMBOSS Pairwise Alignment Algorithms

(Needleman & Wunsch, 1970, supra; Kruskal, 1983, In: Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Sankoff & Kruskal (eds), pp 1 -44, Addison Wesley; programs available from http://www.ebi.ac.uk/tools/emboss/align). All programs may be run using default parameters.

For example, sequence comparisons may be undertaken using the "needle" method of the EMBOSS Pairwise Alignment Algorithms, which determines an optimum alignment (including gaps) of two sequences when considered over their entire length and provides a percentage identity score. Default parameters for amino acid sequence comparisons ("Protein Molecule" option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: Blosum 62. Default parameters for nucleotide sequence comparisons ("DNA Molecule" option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: DNAfull.

In one aspect of the invention, a sequence comparison may be performed over the full length of the reference sequence. As used herein, the term "gene" refers to a DNA sequence from which a polypeptide is encoded or a non-coding, functional RNA is transcribed.

The term "antibiotic resistance gene" encompasses a gene, or the encoding portion thereof, which encodes a product or transcribes a functional RNA that confers antibiotic resistance. For example, the antibiotic resistance gene may be a gene or the encoding portion thereof which contributes to any of the four resistance mechanisms described above. The antibiotic resistance gene may for example encode (1 ) an enzyme which degrades an antibiotic, (2) an enzyme which modifies an antibiotic, (3) a pump such as an efflux pump, or (4) a mutated target which suppresses the effect of the antibiotic.

The term "polynucleotide" refers to a polymeric form of nucleotide of any length, for example RNA (such as mRNA) or DNA. The term also includes, particularly for oligonucleotide markers, the known types of modifications, for example, labels which are known in the art, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as, for example, those with unchanged linkages, e.g., methyl phosphates, phosphotriesters, phosphoamidates, carbamates, etc. and with charged linkages.

The term "polypeptide" as used herein refers to a polymer of amino acids. The term does not refer to a specific length of the polymer, so peptides, oligopeptides and proteins are included within the definition of polypeptide. The term "polypeptide" may include post-expression modifications, for example, glycosylations, acetylations, phosphorylations and the like.

Included within the definition of "polypeptide" are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, unnatural amino acids), polypeptides with substituted linkages, as well as other modifications known in the art both naturally occurring and non-naturally occurring.

The term "microorganism" encompasses prokaryotes such as bacteria and archaea (for example, those belonging to the the Euryarchaeota and Crenarchaeota). Bacteria include both Gram positive and Gram negative bacteria. Some species of clinically significant, pathogenic fungi are included in the definition of microorganisms, for example members of the genus Candida, Aspergillus.. Cryptococcus, Histopiasrna, Pneumocystis and

Stachybotrys.

Various aspects of the invention are shown in the figures and described below. In the present invention, an advantage of using a bacteriophage or conjugative plasmid that comprises the recombinant polynucleotide of the invention is that each serves as a "Trojan horse" that, following infection of the bacteriophage, or following plasmid conjugation, results in the insertion of the "assassin construct" into the target bacteria or other microorganism. The assassin construct once inserted into the target microorganism also provides an immunisation of the cells against the future arrival of a plasmid harbouring antibiotic resistance genes (see Figure 2), in addition, of course to disrupting such genes already present (see Figures 1 and 3).

The assassin constructs then begin the process of degradation of the antibiotic resistance genes. If a DSB created by the Cas DNA-binding protein of the invention destroys a replicon carrying such an antibiotic resistant gene then a microorganism harbouring the antibiotic resistance gene may be killed directly by an assassin contruct. If the microorganism survives the DSB, the resistance gene will be inactivated, and a patient may then be treated with the antibiotic(s) to which the microorganism has now become sensitised.

Importantly, there should be no or reduced direct selection pressure acting against this event if and until patients are subsequently treated with antibiotics. Thus there should be little or no direct selection for bacteriophage (or plasmid) resistance in the pathogenic bacteria or other microorganisms and therefore no or less establishment of an "evolutionary arms race" - sometimes a significant limiting feature of the known use of bacteriophage directly as bactericidal agents.

In the event that DSB-induced killing of a microorganism increases selection pressure for resistance to a bacteriophage or conjugative plasmid delivery agent, the problem could be mitigated by, for example, using a modified Cas DNA polypeptide as defined herein.

This present invention provides potential agents for oral, topical and probiotic, dietary supplement delivery as well as an epidemiological tool to silently inactivate antibiotic resistance genes in pathogenic bacteria or other microorganisms (see Figure 3). Patients scheduled for surgery, or other treatment in hospital, may well be treated with recombinant bacteriophage carrying CRISPR/Cas9 (or other) assassin constructs targeted against antibiotic resistance genes prophylactically in advance of hospital admission. In this way, pathogens present in their microbiome can be directly killed or purged of antibiotic resistance genes in anticipation of any post-operative infection that might occur requiring treatment with antibiotics.

Thus this present invention provides an epidemiological tool to silently inactivate antibiotic resistance genes in pathogenic bacteria. To effect exemplification of the invention, a set of CRISPR/Cas9 assassins targeted against selected antibiotics may be constructed. Variables are the bacteriophage (also referred to herein synonymously simply as "phage"), or conjugative plasmid, or DNA, delivery agent: a range of bacteriophage and plasmid agents may be developed that are specific to a range of important bacterial pathogens.

The extent to which a single generalised bacteriophage or plasmid delivery agent needs to be modified to target different bacterial pathogens depends on the details of the specificity of the interactions of either the phage proteins involved in the phage life-cycle, or the plasmid biology and the pathogenic bacterial target.

Both lysogenic phage that infect hosts and become dormant as prophage, as well as non- virulent phage that replicate but do not kill the host, may be developed. With regards to lysogenic phage specificity, only the lysogenic life cycle of the phage and hence the specificity involved in (i) entry of the phage into the bacterial cell and (ii) its subsequent integration into an attachment site in the target chromosome is required. Integration may not be needed and may be replaced by using the phage to deliver a plasmid that can then excise, for example by cre-lox recombination, and replicate independently in the cell. Non- virulent M13 phage and derivatives thereof may be used.

A functional lytic cycle in lysogenic phage may be retained such that low levels of entry into the lytic cycle in lysogenised bacteria will generate new phage that can go on to

subsequently infect other bacteria (either pathogenic bacteria or non-pathogenic bacteria, to provide immunity). From the point of view of the epidemiological spread of the phage in the pathogenic population this may not be necessary and a single initial infection may suffice. This can be tested experimentally. Optimal conditions for efficient infection and the appropriate multiplicity of infection are identified.

Experiments may be performed in a model system (see Figure 1 ). For example, E. coli carrying multiple drug resistance, for example, to ampicillin, chloramphenicol, and kanamycin or targeted against commonly used antibiotics against which resistance is widespread. Experiments may also be performed with a target pathogen, for example, Klebsiella pneumoniae carbapenemase ("KPC"). KPC is responsible for infection and death in hospitals in the UK.

Delivery by bacteriophage: The phage delivery system (see for example Figure 2, route 2) may be suitable for the treatment of wounds and burns infected by antibiotic-resistant bacteria. Delivery of the assassin construct targeted against these antibiotics by two different E.coli lysogenic bacteriophage: bacteriophage lambda and bacteriophage Mu may be used.

Bacteriophage lambda has a specific integration site in the host chromosome known as the lambda attachment site. Bacteriophage Mu is able to integrate randomly into the host genome and has the advantage that no specific attachment sites in bacteria are required.

Phage may also be used to deliver a plasmid replicon containing the recombinant polynucleotide of the invention that excises by cre-lox recombination following infection as discussed above or by use of phage P1 that replicates as an episome. The specificity involved in successful infection is recognition of a membrane protein on the bacterial cell surface. In the case of lambda this is the maltose permease protein that transports the sugar maltose into the bacterial cell. In the case of bacteriophage Mu the receptor is LPS. Male-specific phage M13 that infect E.coli cells carrying the F-factor plasmid may also be used to deliver CRISPR/Cas9 constructs (or other assassin constructs of the invention) targeted against one or more antibiotic resistance genes. M13 is a well studied phage, which replicates, but does not kill the bacterial host. Successful elimination of resistance to target antibiotics following the lyogenisation by recombinant bacteriophage, or infection by M13 recombinants carrying the assassin construct directed against these antibiotic resistance genes is evaluated.

Studies on other closely related Enterobacteriaceae carrying resistance to the same antibiotics may be performed. These may include Salmonella typhimurium, and Shigella flexneri in addition to Klebsiella pneumoniae discussed above.

In order to do this, modified phage are constructed with different genes encoding the tail fibre protein of the bacteriophage in order to allow it to interact with a different receptor present on the bacterial surface. Lysogenic phage, or male-specific phage, like M13, that are natural hosts of these bacteria may be used.

The phenomenon of phase variation whereby bacteriophage are able to switch their host specificity by the inversion, at a low frequency, of a DNA segment to control gene expression and express alternative tail fibre proteins may also be exploited. And in that way develop phage delivery systems that are versatile. For example phage Mu carries an invertible G segment (regulated by a Mu-encoded site-specific recombinase, Gin) giving rise to two phage types G(+) able to infect E.coli K12 and G(-) able to infect Enterobacter cloacae, Citrobacter freundii Serratia marcescens and Erwinia carotovora.

Another example is the Neisseria gonorrhoeae filamentous phage NgoPhi6, or modified forms thereof. The natural (wild type) phage has a wide host range for Gram negative bacteria (alpha, beta and gamma proteobacteria) - see Piekarowicz et al. (2014 J. Virol. 88: 1002 - 1010). The natural phage is not lytic but lysogenic and has an integration site on the host genome. In one aspect of the invention, the ability of the phage to integrate into the host chromosome is removed and the phage is engineered to replicate independently. The substitution of the left integration site L, CTTATAT with L', CATATAT will eliminate the integration event. In order to replicate the phage genome extra-chromosomally, the M13 ori and M13 gene II can be used to mimic the M13 phage replication. The modification may be kept to a minimum to maintain the ability of the progeny production from the infected bacteria. Although the maximum packaging size is unknown, a phage DNA of around 12 Kb is experimentally demonstrated as packageable and phage progeny produced from the bacteria infected with this phage are infectable. The length of the phage is around 4 microns, which is 4.4 times longer than M13 phage particle, and indicates the higher packaging capacity of DNA longer than M13 - i.e. assuming that the packaging size of DNA is proportional to the size of the phage particle length, DNA packaging capacity of the NgoPhi6 phage will 4 microns/0.9 microns x 6407 nt = 28475 nt, large enough for packaging phage genome along with a recombinant polynucleotide of the invention, including for example a CRISPR-Cas9 construct if required. The exemplified structure of the phage vector

(phagemid) meets the requirements - see Figure 15.

Delivery by conjugative plasmids: Delivery of the assassin constructs targeted against these antibiotics by selected broad-host range conjugative plasmids may be evaluated (see Figure 4, route 3). Here, the plasmids may be delivered by a benign non-pathogenic host. The application here may be for the Gl tract as a probiotic and may be used prophylactically. In this aspect, the possible lethal effects of DSBs caused by Cas DNA-binding protein are not a concern since the plasmid will not be transferred to the recipient.

Similarly demonstration of the successful elimination of resistance to the three target antibiotics following conjugal transfer of plasmids carrying the assassin cargo may be evaluated. Delivery by transformation of linear DNA: Delivery of the assassin construct targeted against these antibiotics by linear double-stranded DNA transformation (see Figure 4, route 1 ) may also be performed. Here, the DNA is delivered via DNA receptor on the surface of the bacteria from the bacterial surrounding environment.

Examples

The present invention is illustrated by the following non-limiting examples.

Example 1

The aim of Example 1 is to demonstrate a proof-of -concept for resurrection of antibiotic efficacy by introduction of a CRISPR/Cas9 construct in non-pathogenic bacterial strains of Escherichia coli. A CRiSPR/Cas9 construct is made directly from genomic DNA isolated from the

Streptococcus pyogenes strain SF370, which is obtained from:

http:/ywwvv.stra!ninfo.net/strains/1 17800/biOwser;jsession!d==A07A638D6D2472EA2FEDBD3 A1928F347.straininfo2. Here the Cas9 coding region plus adjacent regulatory regions and DNA encoding the tracrRNA is extracted by PCR, using sequence-specific DNA primers and cloned into a suitable piasmid cloning vector. A unit repeat of CRISPR array comprising the direct repeats flanking a spacer sequence is similarly extracted by PCR and modified to replace the spacer sequence by a cloning site to allow the subsequent introduction of spacer sequences designed to target DNA regions of choice such as antibiotic resistance genes. It is useful to have a positive selection for bacterial transformants carrying the desired recombinants in which such a spacer sequence has been successfully cloned into the cloning site. Appendix 3 gives an example of such a positive selection.

Alternatively an equivalent CRISPR/Cas9 gene targeting construct, though lacking a positive selection for recombinants, is obtained from a pCas9 piasmid such as for example available from Addgene: http://www.addgene.org/42876/. This piasmid carries the Cas9 gene plus a DNA sequences encoding tracrRNA and CRISPR array with a unique cloning site in order to introduce the spacer sequence desired to target a given DNA sequence for cleavage by the Cas9 endonuclease. For exemplification purposes, use of this existing pCas9 piasmid is described below.

Example 1 shows that bacteria carrying a beta-!actamase (bla) gene conferring resistance to the beta-lactam antibiotic, ampicillin become sensitive to ampicillin following introduction of a modified CRiSPR/Cas9 construct targeted against the Ma gene: the CRISPR/Cas9/anti-i?/a construct. Materials and Methods

A. Bacterial strains, plasmids and phage

1 . Bacterial strains:

DH5a is (F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG O80d/acZAM15 A(lacZYA- a/gr/=)U169, hsdR17(r _K m _K ⁺ ), λ-).

JM109 is (endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB ⁺ A(lac-proAB) e14- [F' traD36 proAB ⁺ lacl ^q lacZAM15] hsdR17(r _K m _K ⁺ )). 2. Plasmids:

pUC18 (Or/ ^' pMB1 ); pCas9 (pACYC184-based vector with Or/ p15A, CRISPR locus plus Cas9 gene, CM ^R ); pCRISPR (On pMB1 , CRISPR, Kn ^R ).

3. Phage: M13mp18

In Example 1 A, in one strain E.coii DH5a and carrying pBR322 a medium copy piasmid or alternatively a low copy piasmid, the CRlSPR/Cas9/anti-£>/a construct is delivered, by naked DNA transformation in piasmid pCas9, designated pCas9::anti-£>/a. The already present piasmid pBR322, or the low copy piasmid expresses the beta-iactarnase derived from bacterial transposon Tn3; pBR322 also carries resistance to tetracycline. As pCas9::anti-d/a carries resistance to chloramphenicol, selection for ceils maintaining pCas9::anti-d/a is achieved by addition of chloramphenicol to the growth medium. In a separate negative control experiment DHSa ceils with pBR332, or a low copy piasmid are transformed with pCas9 (that is a piasmid in all respects like pCas3::anti-£>/a but Iacking anti-d/a which is the spacer sequence targeted against the bla gene: it is predicted that pCas9, in lacking the anis- bia would not be able to attack and inactivate the bla gene. Again pCas9 is maintained by the presence of chloramphenicol.

Beta-!actamase activity can be detected by nitrocefin, which is a chromogenic derivative of cephalosporin, when the beia-lactam ring is hydrolysed, ultraviolet absorption of intact nitrocefin is shifted to around 500 nm, which allows visual detection of the presence of beta lactamase.

Let the total number of bacteria resistant to chloramphenicol be N ₀ and the number of beta- lactamase resistant bacteria be N _r . Growth of N _r manifest as red colonies in the presence of nitrocefin. Let the number of beta lactamase sensitive bacteria be N _w . Growth of N _w colonies manifest as white colonies in the presence of nitrocefin. Thus the efficacy of CRISPR/Cas9/anti-6/a-mediated inactivation of ihe d/a gene efficiency is calculated by measuring the fraction of white colonies, i.e.Nw/N0=(N0-Nr)/N0.

Alternatively beta lactamase activity is seen directly challenging the bacteria on the LB plate containing ampiciliin, Total bacteria resistant to chloramphenicol is N ₀ , ampiciliin resistant bacteria is N _ai then CR!SPR/Cas9/anti-d/a-mediated inactivation of the h!a gene efficiency can be defined by measuring the fraction of the ampiciliin sensitive colony, 1 -Na/N0.

In Example 1 B, an M13 phage delivery system is used to introduce CRiSPR/Cas9/anti-6/a construct into the E, coli strain, JM109, expressing beta lactamase gene from a resident piasmid.

M13::CR!SPR/Cas9/anti-£>/a phage recombinant is prepared as follows: the

CRISPR/Gas9/anti-d/a construct is isolated from pCas9::anti-fe/a by digesting this construct with SaH and Xba\. The digested fragment size is 5796 bp. The fragment is cloned at Sail (GTCGAC) and Xba\ (TCTAGA) site of M13mp18 RF I. The entire size of the M13mp1 8 containing CR!SPR/Cas9/ anti-d/a construct is 13035 bp. This M13 recombinant phage DNA is transformed to E.coli JM109, a bacterial strain carrying the F' piasmid and recombinant M13 phage extruded from the bacteria is purified and used to introduce the CRISPR/Cas9 construct to E.cois JM1 09 harbouring pBR322. As a negative control, an equivalent

M13::CRISPR/Cas9 phage recombinant lacking anti-d/a region is prepared from pCas9 by restriction enzyme Sail and Xbal and the amplicon is cloned at SaH and Xba\ site of M13mp1 8 RF S. When M1 3 infects the bacterial cells, it does not kill them, but the phage DNA replicates inside the cell and expresses phage genes. Thus M13 phage infection with an

M13::CR!SPR/Cas9/anti-£>/a phage recombinant should result in inactivation of the b!a gene, in contrast, in a negative control, to infection by an M13::CRiSPR/Cas9 phage recombinant lacking anti-d/a region. Because M13 phage infection slows down the rate of eel! growth, when infected ceils grown on a lawn of cells yield turbid plaques of slow growing infected cells. If nitrocefin is added to the growth medium, the efficacy of CRI8PR/Cas9/anfi--d/a- mediated inactivation of the d/a gene efficiency is calculated by measuring the fraction of white colonies to red red colonies, when plaques are picked and grown as colonies to remove the background lawn. Plaques may also be picked and plated onto LB plate with/without ampiciliin to score the ratio of ampiciliin sensitive to ampiciliin resistant colonies that will result. B. Selection of spacer sequence from the target sequence

We can use Guide RNA Target Design Tool (see;

https://wwws.blueheronbio.com/external/tools/gRNASrc.jsp) from BlueHeron to select spacer sequence from the target. This program simply returns the 20 nt spacer sequence with the 5 appropriate PAM (protospacer adjacent motif) sequence in the 3' end and GC content. It does not consider secondary structure stability and sequence specificity. Secondary structure prediction and specificity search is performed manually.

We choose the actual spacer sequence from the candidate sequences obtained in the above

10 program, which should meet the following two criteria: 1 ) low tendency to form stable

secondary structure of crRNA, 2) no target DNA on the host genomic DNA. It may be very difficult to find a unique sequence to satisfy criterion No.2. Considering mismatched target data from Figure 3 E in Jinek et al Science 337, 816 (2012), criterion No.2 is relaxed to allow a matched sequence up to the 12th nucleotide position in the target sequence (the first

15 nucleotide position is counted from just next to the PAM sequence). In other words, when the first 12 mer protospacer sequence of the target sequence is completely matched to the 12 mer sequence of crRNA spacer sequence in the 3' end, but the rest of the sequence is not matched, it is assumed that target dsDNA is not cleaved. The specificity check of the protospacer sequence along the E.coli K12 genome sequence is performed by BLAST. The

20 bla sequence is searched against the subject sequence Escherichia coli str. substr. MG1655 (http://www.ncbi. nlm.nih.gov/nucleotide/556503834?report=genbank&log$=nuc lalign&blast_r ank=1 &RID=JUYB76FX014), and each of any matched chromosomal sequence is mapped against the bla sequence for counting the seed sequence from the canonical PAM (NGG) sequence. Secondary structures can be predicted by mFold.

25 (http://mfold.rna. albany.edu/?q=mfold/RNA-Folding-Form) to choose the sequence whose G is large as possible, preferably to be positive for crRNA spacer secondary structure. The following is the way to select the appropriate spacer sequences from the bla sequence.

34

SUBSTITUTE SHEET RULE 26 647- 102

30 666 (F) ACTTTAAAAGTGCTCATCAT TCAT ATCAT None TGG 0.35 -1.30

647- 102

30 666 (F) ACTTTAAAAGTGCTCATCAT 4 5 TGG 0.35 -1.30

767- 103

35 786 (F) TTTACTTTCACCAGCGTTTC GCGTTTC TTTC None TGG 0.43 +1.30

767- 103

35 786 (F) TTTACTTTCACCAGCGTTTC 7 4 TGG 0.43 +2.20

231- 104

65 250 (RC) ATTAATAGACTGGATGGAGG GATGGAGG GAGG GAGG CGG 0.48 +0.00

231- 104

65 250 (RC) ATTAATAGACTGGATGGAGG 8 4 4 CGG 0.48 +0.90

234- 105

66 253 (RC) ACAATTAATAGACTGGATGG GATGG None None AGG 0.39 -0.30

234- 105

66 253 (RC) ACAATTAATAGACTGGATGG 5 AGG 0.39 +0.20

237- 106

67 256 (RC) GCAACAATTAATAGACTGGA GACTGGA CTGGA CTGGA TGG 0.39 -0.20

237- 106

67 256 (RC) GCAACAATTAATAGACTGGA 7 5 5 TGG 0.39 -0.20

241- 107

68 260 (RC) CCCGGCAACAATTAATAGAC AATAGAC AGAC None TGG 0.48 +1.80

241- 107

68 260 (RC) CCCGGCAACAATTAATAGAC 7 4 TGG 0.48 +2.60

259- 108

69 278 (RC) AACTACTTACTCTAGCTTCC AGCTTCC GCTTCC None CGG 0.48 +0.60

259- 108

69 278 (RC) AACTACTTACTCTAGCTTCC 7 6 CGG 0.48 +1.60

284- 109

70 303 (RC) ACGTTGCGCAAACTATTAAC TATTAAC None TAAC TGG 0.43 -1.90

284- 109

70 303 (RC) ACGTTGCGCAAACTATTAAC 7 4 TGG 0.43 -1.90

375- 110

73 394 (RC) TGTAACTCGCCTTGATCGTT TCGTT CGTT CGTT GGG 0.52 -0.50

375- 110

73 394 (RC) TGTAACTCGCCTTGATCGTT 5 4 4 GGG 0.52 +0.10

376- 111

74 395 (RC) ATGTAACTCGCCTTGATCGT TTGATCGT TCGT None TGG 0.48 -0.50

376- 111

74 395 (RC) ATGTAACTCGCCTTGATCGT 8 4 TGG 0.48 +0.10

443- 112

81 462 (RC) AACTTACTTCTGACAACGAT CAACGAT None None CGG 0.43 +0.40

443- 112

81 462 (RC) AACTTACTTCTGACAACGAT 7 CGG 0.43 +0.40

528- 113

84 547 (RC) AGTCACAGAAAAGCATCTTA GCATCTTA None None CGG 0.43 -0.50

528- 113

84 547 (RC) AGTCACAGAAAAGCATCTTA 8 CGG 0.43 +0.50 ess114

90 es? (RC) ACTTTTAAAGTTCTGCTATG GCTATG None None TGG 0.35 -0.70 ess114

90 es? (RC) ACTTTTAAAGTTCTGCTATG 6 TGG 0.35 +0.10

Table 1. Candidate anti-protospacer sequence.

All the target sequence from the bla gene was obtained using Guide RNA Target Design 5 Tool (https://wwws.blueheronbio.com/external/tools/gRNASrc.jsp) from BlueHeron. There are 98 target candidate sequences returned. Bacterial off-target chromosomal short similar

35

SUBSTITUTE SHEET RULE 26 sequences are mapped against the bla gene followed by counting the seed sequence from the canonical PAM sequence. Choose the sequences whose seed sequence number is less than eight and whose Gibbs free energy is relatively large. The summary of the property of the selected target sequences is shown in Table 1. This table also shows nucleotide length of the seed sequence of the off-target sequences on pCas9 and pBR322.

Oligo cassette sequence for spacer sequence

The following four spacer sequences are crRNA generating cassettes targeting beta- lactamase on pBR322 in E. coli as a host strain, which meet the above two criteria. crRNA CR05 cleaves phosphodiester bond between 762nd base C and 763rd base C, CR30 cleaves phosphodiester bond between 198th base G and 199th base A, CR70 cleaves phosphodiester bonds between 575th base T and 576th base A and CR90 cleaves phosphodiester bonds between 221 st base T and 222nd base A on the beta-lactamase gene.

Adaptors for single targets on the beta-lactamase gene.

CR05

5 ' -AAACTAGATAACTACGATACGGGAg SEQ ID NO: 115

atctattgatgctatgccctcAAAA-5' SEQ ID ^' .

CR30

5 ' -AAACACTTTAAAAGTGCTCATCATg SEQ ID NO: 117

tgaaattttcacgagtagtacAAAA-5' SEQ ID NO: 118

Oral

CR70

5 ' -AAACACGTTGCGCAAACTATTAACg SEQ ID NO: 119

tgcaacgcgtttgataattgcAAAA-5 ' SEQ ID NO: 120

Acll

CR90

5 ' -AAACACTTTTAAAGTTCTGCTATGg SEQ ID NO: 121

tgaaaatttcaagacgataccAAAA-5' SEQ ID NO: 122

Oral

Adaptor for dual targets on the beta-lactamase gene.

Internal direct repeat sequence is italicised and underlined.

CR30+CR90 SEQ ID NO: 123

5' -ARACACTTTARAAGTGCTCATCATGrrrrAGAGCrArGCrGrrrrGAarGGTCCCAaAa CaCTTTTAAAGTTCTGCTATGg tgaaattttcacgagtagtacaaaatctcgatacgacaaaacttaccagggttttgtgaa aatttcaagacgataccAAAA Oral CR30 Oral CR90

SEQ ID NO: 124

36

SUBSTITUTE SHEET RULE 26 The 5' end of each oligo is phosphorylated and ready for cloning at Bsa\ sites.

Sites of six base cutter restriction endonucleses are underlined, which are useful to screen the recombinants. We can also employ one of the cassette oligos as a primer to screen the recombinants by PCR together with another unique primer for the plasmid vector.

Appendix 1

pCas9 plasmid sequence (SEQ ID NO: 125)

Cas 9 gene, CRISPR expression locus and tracrRNA (all from S. pyogenes)

GAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAA CTTGTGCTTATTTTTCTTTACGG TCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTG ACTGAAATGCCTCAAAATGT TCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATT TTAGCTTCCTTAGCTCCTGA AAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTT GGAACCTCTTACGTGCCGAT CAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAG GATTTATTTATTCTGCGAAG TGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTA CTGATTTAGTGTATGATGGT GTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTG ACGGGGTGGTGCGTAACGGC AAAAGCACCGCCGGACATCAGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTG ATGAGGGTGTCAGTGAAGTG CTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGA TATATTCCGCTTCCTCGCTC ACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGC GGAGATTTCCTGGAAGATGC CAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTC CGCCCCCCTGACAAGCATCA CGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC GTTTCCCCCTGGCGGCTCCC TCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGC GTTTGTCTCATTCCACGCCT GACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGT TCAGTCCGACCGCTGCGCCT TATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAG CAGCCACTGGTAATTGATTT AGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGG TGACTGCGCTCCTCCAAGCC AGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGG CGGTTTTTTCGTTTTCAGAG CAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAGATAAAA TATTTCTAGATTTCAGTGCA ATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTGTAATTCTC ATGTTTGACAGCTTATCATC GATAAGCTTTAATGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGT ATGAAATCTAACAATGCGCT CATCGTCATCCTCGGCACCGTCACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGT ACTGCCGGGCCTCTTGCGGG ATTACGAAATCATCCTGTGGAGCTTAGTAGGTTTAGCAAGATGGCAGCGCCTAAATGTAG AATGATAAAAGGATTAAGAG ATTAATTTCCCTAAAAATGATAAAACAAGCGTTTTGAAAGCGCTTGTTTTTTTGGTTTGC AGTCAGAGTAGAATAGAAGT ATCaaaaaaagcaccgactcggtgccactttttcaagttgataacggactagccttattt taacttgctatgctgttttg aatggttccAACAAGATΎΑΎΎΎΎΑΤΑΑΟΤΤΤΤΑΤΑΑCAAAT AATCAAGGAGAAATTCAAAGAAATTTATCAGCCATAAAA CAATACTTAATACTATAGAATGATAACAAAATAAACTACTTTTTAAAAGAATTTTGTGTT ATAATCTATTTATTATTAAG TATTGGGTAATAT τTTTTGAAGAGATATTTTGAAAAAGAAAAATTAAAGCATATTAAACTAATTTCGGAGGT CATTAAAA CTATTATTGAAATCATCAAACTCATTATGGATTTAATTTAAACTTTTTATTTTAGGAGGC AAAA

ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCG GTGATCACTGATGAATATAAGGT TCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCT TATAGGGGCTCTTTTATTTG ACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACAC GTCGGAAGAATCGTATTTGT TATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGA CTTGAAGAGTCTTTTTTGGT GGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGC TTATCATGAGAAATATCCAA CTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAA TCTATTTGGCCTTAGCGCAT ATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGAT GTGGACAAACTATTTATCCA GTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGA TGCTAAAGCGATTCTTTCTG CACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGA AAAATGGCTTATTTGGGAAT CTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAA GATGCTAAATTACAGCTTTC AAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGC TGATTTGTTTTTGGCAGCTA AGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTA AGGCTCCCCTATCAGCTTCA ATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGA CAACAACTTCCAGAAAAGTA TAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGC TAGCCAAGAAGAATTTTATA AATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAA ATCGTGAAGATT GCTGCGC AAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCAT GCTATTTTGAGAAGACAAGA AGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCG AATTCCTTATTATGTTGGTC

37

SUBSTITUTE SHEET RULE 26 CATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTA CCCCATGGAATTTTGAAGAA GTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAA AATCTTCCAAATGAAAAAGT ACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGT CAAATATGTTACTGAAGGAA TGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCA AAACAAATCGAAAAGTAACC GTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATT TCAGGAGTTGAAGATAGATT TAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTT GGATAATGAAGAAAATGAAG ATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGG AAAGACTTAAAACATATGCT CACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGA CGTTTGTCTCGAAAATTGAT TAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGG TTTTGCCAATCGCAATTTTA TGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGT CTGGACAAGGCGATAGTTTA CATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACT GTAAAAGTTGTTGATGAATT GGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAA TCAGACAACTCAAAAGGGCC AGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTC AGATTCTTAAAGAGCATCCT GTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGA GACATGTATGTGGACCAAGA ATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCT TAAAGACGATTCAATAGACA ATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAG AAGTAGTCAAAAAGATGAAA AACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTA ACGAAAGCTGAACGTGGAGG TTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAAT CACTAAGCATGTGGCACAAA TTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTA AAGTGATTACCTTAAAATCT AAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAAT TACCATCATGCCCATGATGC GTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGA GTTTGTCTATGGTGATTATA AAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCG CAAAATATTTCTTTTACTCT AATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGC CCTCTAATCGAAACTAATGG GGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATT GTCCATGCCCCAAGTCAATA TTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAA GAAATTCGGACAAGCTTATT GCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCT TATTCAGTCCTAGTGGTTGC TAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCAC AATTATGGAAAGAAGTTCCT TTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACT TAATCATTAAACTACCTAAA TATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTA CAAAAAGGAAATGAGCTGGC TCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGG TAGTCCAGAAGATAACGAAC AAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCA GTGAATTTTCTAAGCGTGTT ATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAA CCAATACGTGAACAAGCAGA AAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTT TGATACAACAATTGATCGTA AACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTG GTCTT ATGAAACACGCATT GATTTGAGTCAGCTAGGAGGTGACTGA

-10 promoter (SEQ ID NO: 127)

AGTATATTTTAGATGAAGATTATTTCTTAATAACTAAAAATATGGTATAATACTCTT ¾3¾¾?8 ¾1 Leader sequence (SEQ ID NO: 128)

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^

Direct repeat Spacer Direct

|¾|GTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAACTGAGACCAGTCTCGGAA GCTCAAAGGTCTCGTTTTAGAGCT

Bsa I Bsa I repeat Spacer Direct repeat

ATGCTGTTTTGAATGGTCCCAAAACTTCAGCACACTGAGACTTGTTGAGTTCCATGT TTTAGAGCTATGCTGTTTTGAAT

GGACTCCATTCAACATTGCCGATGATAACTTGAGAAAGAGGGTTAATACCAGCAGTC GGATACCTTCCTATTCTTTCTGT TAAAGCGTTTTCATGTTATAATAGGCAAAAGAAGAGTAGTGTGArCGrCCArrCCGACAG CArCGCCAGrCACrArGGCG TGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGT CCGACCGCTTTGGCCGCCGC CCAGTCCTGCTCGCTTCGCTACTTGGAGCCACTATCGACTACGCGATCATGGCGACCACA CCCGTCCTGTGGATCCTCTA CGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATAT CGCCGACATCACCGATGGGG AAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAG GCCCCGTGGCCGGGGGACTG TTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAAC CTACTACTGGGCTGCTTCCT AATGCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCCCTTGAGAGCCTTCAACCCAGT CAGCTCCTTCCGGTGGGCGC GGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGAC AGGTGCCGGCAGCGCTCTGG GTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCG GTATTCGGAATCTTGCACGC CCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCAT TATCGCCGGCATGGCGGCCG ACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTA TGATTCTTCTCGCTTCCGGC GGCA CGGGATGCCCGCGTTGCAGGCCA GCTGTCCAGGCAGGTAGA GACGACCA CAGGGACAGCTTCAAGGA CGCT CGCGGCTCTTACCAGCCTAACTTCGATCATTGGACCGCTGATCGTCACGGCGATTTATGC CGCCTCGGCGAGCACATGGA

38

SUBSTITUTE SHEET RULE 26 ACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTC GCGGTGCATGGAGCCGGGCC ACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTG GAGCCAATCAATTCTTGCGG AGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCC AGCAGCCGCACGCGGCGCAT CTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGAC CCGGCTAGGCTGGCGGGGTT GCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCT GCAAAACGTCTGCGACCTGA GCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCC CCTACGTGCTGCTGAAGTTG CCCGCAACAGAGAGTGGAACCAACCGGTGATACCACGATACTATGACTGAGAGTCAACGC CATGAGCGGCCTCATTTCTT ATTCTGAGTTACAACAGTCCGCACCGCTGTCCGGTAGCTCCTTCCGGTGGGCGCGGGGCA TGACTATCGTCGCCGCACTT ATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCCCAACAGTCCC CCGGCCACGGGGCCTGCCAC CATACCCACGCCGAAACAAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATG CTGCTGGCTACCCTGTGGAA CACCTACATCTGTATTAACGAAGCGCTAACCGTTTTTATCAGGCTCTGGGAGGCAGAATA AATGATCATATCGTCAATTA TTACCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCA CACTGCTTCCGGTAGTCAAT AAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACG ACCGGGTCGAATTTGCTTTC GAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCACCAGGCGTTTAAG GGCACCAATAACTGCCTTAA AAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTG CCGACATGGAAGCCATCACA GACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATA TTTGCCCATGGTGAAAACGG GGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGG GATTGGCTGAGACGAAAAAC ATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCT TGCGAATATATGTGTAGAAA CTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATG GAAAACGGTGTAACAAGGGT GAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACG

The reverse complement of tracrRNA is in lowercase, Cas9 coding sequence (SEQ ID NO: 126) is boxed and direct repeat sequence in CRISPR is in bold. Promoter sequences were predicted by neural network algorithm (http://www.fruitfly.org/seq_tools/promoter.html). The two unique sites, Sal I (GTCGAC) and 6a I (TCTAGA) highlighted in bold italicised black are utilised to isolate the CRISPR/Cas9 construct for cloning into M13mp18. The pACYC184 backbone sequence is italicised.

The information of Appendix 1 above is alternatively presented as follows. pCas9 plasmid sequence (SEQ ID NO: 125)

Cas 9 gene, CRISPR expression locus and tracrRNA (all from S. pyogenes)

GAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTT GTGCTTATTTTTCTTTACGG TCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTG ACTGAAATGCCTCAAAATGT TCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATT TTAGCTTCCTTAGCTCCTGA AAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTT GGAACCTCTTACGTGCCGAT CAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAG GATTTATTTATTCTGCGAAG TGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTA CTGATTTAGTGTATGATGGT GTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTG ACGGGGTGGTGCGTAACGGC AAAAGCACCGCCGGACATCAGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTG ATGAGGGTGTCAGTGAAGTG CTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGA TATATTCCGCTTCCTCGCTC ACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGC GGAGATTTCCTGGAAGATGC CAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTC CGCCCCCCTGACAAGCATCA CGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC GTTTCCCCCTGGCGGCTCCC TCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGC GTTTGTCTCATTCCACGCCT GACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGT TCAGTCCGACCGCTGCGCCT TATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAG CAGCCACTGGTAATTGATTT AGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGG TGACTGCGCTCCTCCAAGCC AGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGG CGGTTTTTTCGTTTTCAGAG CAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAGATAAAA TATTTCTAGATTTCAGTGCA ATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTGTAATTCTC ATGTTTGACAGCTTATCATC GATAAGCTTTAATGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGT ATGAAATCTAACAATGCGCT CATCGTCATCCTCGGCACCGTCACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGT ACTGCCGGGCCTCTTGCGGG AI ACGAAATCATCCTGTGGAGCTTAGTAGGTTTAGCAAGATGGCAGCGCCTAAATGTAGAAT GATAAAAGGATTAAGAG ATTAATTTCCCTAAAAATGATAAAACAAGCGTTTTGAAAGCGCTTGTTTTTTTGGTTTGC AGTCAGAGTAGAATAGAAGT KTCAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTT TAACTTGCTATGCTGTTTTG AATGGTTCCRRCRRGKnRttttataacTtttataacaaataatcaaggagaaattcaaag aaatttatCRGCCKTRRRR

CAATACTTAATACTATAGAATGATAACAAAATAAACTACTTTTTAAAAGAATTTTGT GTTATAATCTATTTATTATTAAG TATtgggtaatattttttgaagagatattttgaaaaagaaaaaTtaaagcataTTAAACT AATTTCGGAGGTCATTAAAA

CTATTATTGAAATCATCAAACTCATTATGGATTTAATTTAAACTTTTTATTTTAGGA GGCAAAAATGGATAAGAAATACT

39

SUBSTITUTE SHEET RULE 26 CAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATA AGGTTCCGTCTAAAAAGTTC AAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTA TTTGACAGTGGAGAGACAGC GGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTAT TTGTTATCTACAGGAGATTT TTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTT TGGTGGAAGAAGACAAGAAG CATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATAT CCAACTATCTATCATCTGCG AAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGC GCATATGATTAAGTTTCGTG GTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTA TCCAGTTGGTACAAACCTAC AATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTT TCTGCACGATTGAGTAAATC AAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGG GAATCTCATTGCTTTGTCAT TGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGC TTTCAAAAGATACTTACGAT GATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCA GCTAAGAATTTATCAGATGC TATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGC TTCAATGATTAAACGCTACG ATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAA AGTATAAAGAAATCTTTTTT GATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTT TATAAATTTATCAAACCAAT TTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCT GCGCAAGCAACGGACCTTTG ACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGAC AAGAAGACTTTTATCCATTT TTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTT GGTCCATTGGCGCGTGGCAA TAGTCGTTTTGCA GGA GAC CGGAAGTCTGAAGAAACAATTACCCCATGGAATTT GAAGAAGT GTCGATAAAGG G CTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAA AAGTACTACCAAAACATAGT TTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAA GGAATGCGAAAACCAGCATT TCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGT AACCGTTAAGCAATTAAAAG AAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATA GATTTAATGCTTCATTAGGT ACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAAT GAAGATATCTTAGAGGATAT TGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATA TGCTCACCTCTTTGATGATA AGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAAT TGATTAATGGTATTAGGGAT AAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAAT TTTATGCAGCTGATCCATGA TGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAG TTTACATGAACATATTGCAA ATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATG AATTGGTCAAAGTAATGGGG CGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAG GGCCAGAAAAATTCGCGAGA GCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCA TCCTGTTGAAAATACTCAAT TGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACC AAGAATTAGATATTAATCGT TTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATA GACAATAAGGTCTTAACGCG TTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGAT GAAAAACTATTGGAGACAAC TTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTG GAGGTTTGAGTGAACTTGAT AAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCA CAAATTTTGGATAGTCGCAT GAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAA ATCTAAATTAGTTTCTGACT TCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATG ATGCGTATCTAAATGCCGTC GTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGAT TATAAAGTTTATGATGTTCG TAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTA CTCTAATATCATGAACTTCT TCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTA ATGGGGAAACTGGAGAAATT GTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTC AATATTGTCAAGAAAACAGA AGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCT TATTGCTCGTAAAAAAGACT GGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGG TTGCTAAGGTGGAAAAAGGG AAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGT TCCTTTGAAAAAAATCCGAT TGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACC TAAATATAGTCTTTTTGAGT TAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGC TGGCTCTGCCAAGCAAATAT GTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAAC GAACAAAAACAATTGTTTGT GGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCG TGTTATTTTAGCAGATGCCA ATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAG CAGAAAATATTATTCATTTA TTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGAT CGTAAACGATATACGTCTAC AAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACG CATTGATTTGAGTCAGCTAG GAGGTGACTGAAGTATattttagatgaagattatttcttaataactaaaaatatggTata atactcTTAaTAAaTGCAGT AATACAGGGGCTTTTCAAGACTGAAGTCTAGCTGAGACAAATAGTGCaATTACGAAATTT TTTAGACAAAAATAGTCTAC GAGGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAACTGaGaCC¾GTCTCGG¾AGC TC¾AAGGrcrCGTTTTAGAGCT ATGCTGTTTTGAATGGTCCCAAAACTTCAGCACACTGAGACTTGTTGAGTTCCATGTTTT AGAGCTATGCTGTTTTGAAT GGACTCCATTCAACATTGCCGATGATAACTTGAGAAAGAGGGTTAATACCAGCAGTCGGA TACCTTCCTATTCTTTCTGT TAAAGCGTTTTCATGTTATAATAGGCAAAAGAAGAGTAGTGTGATCGrCCArrCCGACAG CATCGCCAGrCACrarGGCG TGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGT CCGACCGCTTTGGCCGCCGC CCAGTCCTGCTCGCTTCGCTACTTGGAGCCACTATCGACTACGCGATCATGGCGACCACA CCCGTCCTGTGGATCCTCTA CGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATAT CGCCGACATCACCGATGGGG AAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAG GCCCCGTGGCCGGGGGACTG TTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAAC CTACTACTGGGCTGCTTCCT AATGCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCCCTTGAGAGCCTTCAACCCAGT CAGCTCCTTCCGGTGGGCGC GGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGAC AGGTGCCGGCAGCGCTCTGG GTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCG GTATTCGGAATCTTGCACGC CCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCAT TATCGCCGGCATGGCGGCCG ACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTA TGATTCTTCTCGCTTCCGGC GGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAG GGACAGCTTCAAGGATCGCT CGCGGCTCTTACCAGCCTAACTTCGATCATTGGACCGCTGATCGTCACGGCGATTTATGC CGCCTCGGCGAGCACATGGA ACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTC GCGGTGCATGGAGCCGGGCC ACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTG GAGCCAATCAATTCTTGCGG AGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCC AGCAGCCGCACGCGGCGCAT CTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGAC CCGGCTAGGCTGGCGGGGTT GCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCT GCAAAACGTCTGCGACCTGA GCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCC CCTACGTGCTGCTGAAGTTG CCCGCAACAGAGAGTGGAACCAACCGGTGATACCACGATACTATGACTGAGAGTCAACGC CATGAGCGGCCTCATTTCTT ATTCTGAGTTACAACAGTCCGCACCGCTGTCCGGTAGCTCCTTCCGGTGGGCGCGGGGCA TGACTATCGTCGCCGCACTT ATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCCCAACAGTCCC CCGGCCACGGGGCCTGCCAC CATACCCACGCCGAAACAAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATG CTGCTGGCTACCCTGTGGAA CACCTACATCTGTATTAACGAAGCGCTAACCGTTTTTATCAGGCTCTGGGAGGCAGAATA AATGATCATATCGTCAATTA TTACCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCA CACTGCTTCCGGTAGTCAAT AAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACG ACCGGGTCGAATTTGCTTTC GAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCACCAGGCGTTTAAG GGCACCAATAACTGCCTTAA

40

SUBSTITUTE SHEET RULE 26 Backbone vector pACYC184 sequence is italicised, sequence positions are numbered from G of EcoRI site underlined. The reverse complement of tracrRNA is in italicised bold located from 1844 to 1929, Cas9 initiation and termination codons are indicated by bold three letters, starting at nucleotide No. 2225 and ending at 6331 followed by leader sequence 6389-6483 indicated by italicised bold letters, first, second and third direct repeat sequences are underlined, between the first and second direct repeat in which spacer cloning region is located.

This spacer cloning region contains two inverted Bsa\ sites indicated by bold italicised letters 5'-GAGACC-3' and 5'-GGTCTC-3' for creating 5' four bases protruding spacer cloning sites 5'-GTTT-3' and 5'-TTTT-3', respectively. Promoter sequences were predicted as above, indicated by lower case for forward promoter for Cas9 and leader sequence and italicised lower case for reverse promoter for tracrRNA, the putative transcription start site is indicated by bold uppercase. The two unique sites, Sal I (GTCGAC) and 6a I

(TCTAGA) highlighted in bold italicised black are utilised to isolate the CRISPR/Cas9 construct for cloning into M13mp18.

Appendix 2

Modification of spacer cloning site on pCas9

This system exploits the fact that expression of a minigene yields a certain oligopeptide that depletes the tRNA pool in the host cell, when induced for expression, which leads to the disruption of protein synthesis (Tenson T, Vega-Herrera J, Kloss P, Guarneros G, Mankin AS. J. Bacteriol. 1999; 181 :1617-1622). Applying this concept to cloning the spacer sequence: when the spacer sequence is inserted this interrupts the minigene and the oligopeptide expression does not occur, thus tRNA is not depleted, thus only the bacteria harbouring a plasmid with insert can survive under conditions of induced expression; whilst bacteria harbouring the plasmid without insert cannot grow. The following (SEQ ID NO: 129) is the sequence structure. In order to construct this structure, bacteria should express the lac I repressor constitutively to switch off minigene

transcription. Recombinant selection should be performed in the presence of IPTG to induce transcription.

41

SUBSTITUTE SHEET RULE 26 5' -AAAACTGAGAcc mm s ^ w« ATTAGGCAccccAGGc e M MMSMmmwMi^&

Bsal 031ac -35 Ptac -10

TGTG α^Μ ^^β&^^^^^ΐ CACAC|GGAGG|TCACATATGAGA GAATT c;||tl l||| tlf 111

021ac S.D. M R Trp terminator

Bsal

Bsa\ recognition sites are underlined, lac operator 3 and 1 are bold italicised. Tac promoter is in bold letters, -35 and -10 region in the promoter sequence are underlined, Shine- Dalgarno sequence is boxed, first dipeptide is in bold M and R followed by three consecutive termination codons italicised TAA. Tryptophan terminator signal sequence is employed, indicated by italicised letters.

Appendix 3

Expected cleavage site on target sequence using CR90 spacer sequence

First processing event

tracrRNA hybridises to the direct repeat region of pre-crRNA indicated by upper case letters using the sequence underlined. Bacterial RNase III cleaves the double-stranded RNA region at indicated position "|", first processing event.

tracrRNA 3' -uuuuuuucguggcu

('SEQID NO: 130)' aaaaguggcaccga ⁹

a

guucaacuauugccuga

u

⁵ ' -gga-acca uca I aaacagcauagcaaguuaaaauaaggc

CAAACCCUGGUAA I GUUUUGTCGUAUCG AGAUUUUG-5' pre-crRNA a

(SEQID NO: 131) cuuuuaaaguucugcuajgGUUUUAGAGCUAUGCTGUUUUG | AAUGGUCCCAAAAC-3 '

uaaaauugaacgauacgacaaaJ acu accaagg-5' acggugaaaaaguucaacuauugccugaucggaa

g

ccgaucggugcuuuuuuu-3' tracrRNA (SEQ ID NO: 130)

42

SUBSTITUTE SHEET RULE 26 Structure after 1 st processing event

(SEQ ID NO: 132)

5' -ggaaoeauuca

CAAACCCUGGUAA

pre-crRNA a

cuuuuaaaguucugcuaugG|t|i !Ij¾GA GCUAUGCTGUUUUG

aaaa ugaacgauacgacaaa

agccacggugaaaaaguucaacuauugccugaucggaa

g

ucggugcuuuuuuu-3' tracrRNA

The first processing event may also be depicted as follows: tracrRNAs indicated by lower case letters hybridise to the direct repeat region of pre-crRNA indicated by upper case bold black letters. Bacterial RNase III cleaves the double-stranded RNA region at indicated position with arrows, which is first processing event. Phosphodiester bond between 22 ^nd and 23 ^rd base in the first and second direct repeat are cleaved. Italicised spacer sequence CR90 is boxed.

43

SUBSTITUTE SHEET RULE 26 Second processing event

The 2nd cleavage point ( ^* ) is around 20 nt away from the 3' end of the spacer sequence. "Note that the 2nd processing event occurs at a specific distance from the 1 st cleavage within the repeats. Considering that spacer sequences are not identical among each other, it is thus likely that the 2nd processing event within the spacers is distance-dependent rather than sequence-dependent" (see Supplementary figure 2 legend in Nature. Mar 31 , 201 1 ;471 (7340):602-607). Although the above-referenced article does not particularly specify the enzyme and/or mechanism involved in this 2nd processing event, it is most likely Cas9 is involved in this 2nd cleavage. Considering this cleavage position within the spacer sequence, the whole spacer sequence is not contributing to the target recognition, instead only 20 nt spacer sequence is utilised for hybridisation.

(SEQID NO: 132)

5 ' -ggaaccauuca

*CAAACCCUGGUAA-5 ' pre-crRNA ' ^{SEQ ID NO: 34} >

a

cuuuuaaaguucugcuaugGUUUUAGA GCUAUGCTGUUUUG-3 '

uaaaauugaacgauacgacaaa-5'

agccacggugaaaaaguucaacuauugccugaucggaa

g

ucggugcuuuuuuu-3' tracrRNA (SEQ ID NO: 133)

The second processing event may also be depicted as follows: The 2nd cleavage point indicated by arrows around 20 nt away from the 3' end of the spacer sequence in the above figure.

The above note quoted from Nature, and our comment, applies.

The italicised spacer sequence CR90 is in box.

44

SUBSTITUTE SHEET RULE 26 -A

CUUUUAAAGUUCUGCUAU/pGUUUUAGA GCUAUGCTGUUUUG-3 ' (SEQ ID NO: 135)

o66eerveeeerui6eeo6erveo6eoeee- , c

n ³

eBnooBnrvenoeeormB

⁵ no66n6onnnnnnn- , _ε (SEQ ID NO: 133)

Structure after 2nd processing event

Target DNA cleavage

The part of the target beta-lactamase DNA sequence containing CR90 is shown. crRNA hybridises to its complementary sequence of the target region, Cas9 cleavage points are indicated by dot "." and the PAM sequence tgg is indicated in the box.

agccacggugaaaaaguucaacuauugccugaucggaa

g

ucggugcuuuuuuu - 3 ' tracrKNA

An alternative depiction of the target DNA cleavage is shown below. Here, the part of the target beta-lactamase DNA sequence containing CR90 proto-spacer sequence is shown in lower case bold black letters. crRNA hybridises to its complementary sequence of the proto-spacer strand, Cas9 cleavage points are indicated by dot arrows and the PAM sequence tgg is under lined.

45

SUBSTITUTE SHEET RULE 26 acttttaaagttctgctatg

5 ' ... ccaagtca tggcgcggta ...3 '

3' ...ggttcagt accgcgccat...5' (SE ^Q ID NO: 135) tgaaaatttcaagacgatac ^—"^" ^{^}

5 '-ACUUUUAAAGUUCUGCUAUGGUUUUAGA GCUAUGCTGUUUUG-3 '

oB&BBtlBBBBtinBBBoBBtlBoBBOBBB- ,q (SE _Q ID NO- 133) eBnooBnrvenoeeoririB

5ηοββηβοηηηηηηη- _{; ε}

Annealing to proto-spacer DNA and cleavage points

Example 2

The following experiments describe some proof-of-concept experiments performed to demonstrate that the CRISPR-Cas9 system can be used to inactivate antibiotic resistance in bacteria. They describe the construction of a generally applicable DNA cassette, described in the Examples to deliver the CRISPR-Cas9 system plus a derivative carrying a spacer sequence targeted against an antibiotic resistance gene for delivery by naked DNA transformation and bacteriophage infection and also to demonstrate inhibition of the spread of antibiotic resistance by plasmid conjugation.

Construction of pNB100

pNB100 is a vector to express the CRISPR-Cas9 system in E.coli with the appropriate unique restriction site, Bsa I, to clone any desired spacer sequence between two direct repeats in the CRISPR locus. The backbone of the vector is derived from pACYC184 and the CRISPR-cas9 locus is inserted into Eco RV site of the vector. Three regions of the CRISPR-cas9 locus were amplified by PCR from the genomic DNA of Streptococcus pyogenes strain SF370, purchased from the ATCC, and assembled by Gibson assembly (Gibson DG, et.al. Nature Methods 2009; 6: 343-345) along with the pACYC184 vector in the reaction. The sequence of the final construct was verified by Sanger sequencing. The CRISPR-Cas9 activity was confirmed using a derivative of pNB100, pNB102, carrying a spacer sequence targeted against the beta lactamase genes of the bacterial transposons Tn3 and Tn1.

46

SUBSTITUTE SHEET RULE 26 The amplified sequence of the three regions and amplicon image on the gel

The following sequences are the three regions amplified by PCR. Underlined sequences are template-specific primer sequences, bold letters are overlapping sequences used for Gibson assembly.

1 . Fragment 1 (SEQ ID NO: 140), tracrRNA-cas9 : amplicon length = 4758 bp

Forward primer is from 854170 to 854193 and reverse primer is from 858867 to 858848 on

S. pyogenes SF370 genomic DNA.

ATGCCGGTACTGCCGGGCCTCTTGCGGGATCCAGAAGTCTTTTTCTTGCACTGTTTC CTTTTCTTTATGATAGTTTAC GAAAT C AT C C TGTGGAGCTTAGTAGGTTTAGCAAGATGGCAGCGCCTAAATGTAGAATGATAAAAGGATT AAGAGATTAATTTCCCTAAAAATGATAA AACAAGCGTTTTGAAAGCGCTTGTTTTTTTGGTTTGCAGTCAGAGTAGAATAGAAGTATC AAAAAAAGCACCGACTCGGTGCCACTTT TTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATGCTGTTTTGAATGGTTCCAA CAAGATTATTTTATAACTTTTATAACAA AT AAT C AAGGAGAAAT T C AAAGAAAT T T AT C AGC CAT AAAAC AAT AC T T AAT AC TAT AGAAT GAT AAC AAAAT AAAC T AC T T T T T AAA AGAATTTTGTGTTATAATCTATTTATTATTAAGTATTGGGTAATATTTTTTGAAGAGATA TTTTGAAAAAGAAAAATTAAAGCATATT AAACTAATTTCGGAGGTCATTAAAACTATTATTGAAATCATCAAACTCATTATGGATTTA ATTTAAACTTTTTATTTTAGGAGGCAAA AATGGATAA GAAAT ACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAAT AT AAGGTTCCGTCT AAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGG GCTCTTTTATTTGACAGTGGAGAGACAG CGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTA TTTGTTATCTACAGGAGATTTTTTCAAA TGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGA AGAAGACAAGAAGCATGAACGTCATCCT ATTTTTGGAAAT AT AGTAGATGAAGTTGCTTAT CAT GA GAAAT ATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTG ATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTC ATTTTTTGATTGAGGGAGATTTAAATCC TGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATT TGAAGAAAACCCTATTAACGCAAGTGGA GTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTC ATTGCTCAGCTCCCCGGTGAGAAGAAAA ATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAA ATTTTGATTTGGCAGAAGATGCTAAATT ACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGA TCAATATGCTGATTTGTTTTTGGCAGCT AAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAAT ACT GAAAT AACTAAGGCTCCCCTATCAGCTTCAATGATTA AACGCTACGAT GAAC AT CATCAAGACTTGACTCTTT T AAAAGC T T T AGT T C GAC AAC AAC T T C CAGAAAAGT AT AAAGAAAT C T T T T T TGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATT TTATAAATTTATCAAACCAATTTTAGAA AAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAG CAACGGACCTTTGACAACGGCTCTATTC CCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATC CATTTTTAAAAGACAATCGTGAGAAGAT TGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAG TCGTTTTGCATGGATGACTCGGAAGTCT GAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAA TCATTTATTGAACGCATGACAAACTTTG ATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTA CGGTTTATAACGAATTGACAAAGGTCAA ATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCAT TGTTGATTTACTCTTCAAAACAAATCGA AAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGT GTTGAAATTTCAGGAGTTGAAGATAGAT TTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTT TGGATAATGAAGAAAATGAAGATATCTT AGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACT TAAAACATATGCTCACCTCTTTGATGAT AAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAA TTGATTAATGGTATTAGGGATAAGCAAT CTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGC AGCTGATCCATGATGATAGTTTGACATT TAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATAT TGCAAATTTAGCTGGTAGCCCTGCTATT AAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGG CATAAGCCAGAAAATATCGTTATTGAAA TGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAAC GAATCGAAGAAGGTATCAAAGAATTAGG AAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTA TCTCTATTATCTCCAAAATGGAAGAGAC ATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATT GTTCCACAAAGTTTCCTTAAAGACGATT CAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTC CAAGTGAAGAAGTAGTCAAAAAGATGAA AAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTT AACGAAAGCTGAACGTGGAGGTTTGAGT GAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAG CATGTGGCACAAATTTTGGATAGTCGCA TGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAA AATCTAAATTAGTTTCTGACTTCCGAAA AGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTA TCTAAATGCCGTCGTTGGAACTGCTTTG ATTAA GAAAT ATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAA TGATTGCTAAGTCTGAGC AA GAAAT AGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAAT AT CAT GAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGA GATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAA AGGGCGAGATTTTGCCACAGTGCGCAAA GTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTC TCCAAGGAGTCAATTTTACCAAAAAGAA ATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTG ATAGTCCAACGGTAGCTTATTCAGTCCT AGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACT AGGGATCACAATTATGGAAAGAAGTTCC TTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGAC TTAATCATTAAACTACCTAAATATAGTC TTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAG GAAATGAGCTGGCTCTGCCAAGCAAATA TGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAA CGAACAAAAACAATTGTTTGTGGAGCAG CATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATT TTAGCAGATGCCAATTTAGATAAAGTTC TTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTC ATTTATTTACGTTGACGAATCTTGGAGC TCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAA AGAAGTTTTAGATGCCACTCTTATCCAT CAAT CCATCACTGGTCTTTAT GAAAC AC GC AT T GAT T T GAGTCAGCTAGGAGGTGACTGATGGCCACGTGAACTATATGATTTTCCGC AGTATA

2. Fragment 2 (SEQ ID NO: 141 ), Leader and first direct repeat : amplicon length = 276 bp Forward primer is from 860648 to 860671 and reverse primer is from 860862 to 860806 on S. pyogenes genomic DNA.

ATTGATTTGAGTCAGCTAGGAGGTGACTGATGGCC AC GT GAAC TAT AT GAT T T T C C G CAGT AT AT T T T AGAT GAAGAT TATTTCTTAA TAACTAAAAATATGGTATAATACTCTTAATAAATGCAGTAATACAGGGGCTTTTCAAGAC TGAAGTCTAGCTGAGACAAATAGTGCGA T TAC GAAAT T T T T TAGACAAAAATAGTCTACGAGGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAACTGA GACCAGTCTCGGACG TCCAAAGGTCTC

3. Fragment 3 (SEQ ID NO: 144), Second direct repeat: amplicon length = 452 bp

Forward primer is from 861221 to 861276 and reverse primer is from861613 to 861594 on S. pyogenes genomic DNA. Decamer sequence from 861246-861255 GGTCTCCATT (SEQ ID NO: 142), which contains Bsa\ recognition sequence on the genomic DNA, was substituted with GGTCCCAAAA (SEQ ID NO: 143) to destroy Bsa\ recognition sequence and convert the 7th truncated direct repeat in the CRISPR array on the genome to the canonical 2nd direct repeat sequence in this vector.

GAGACCAGTCTCGGACGTCCAAAGGTCTCGTTTTAGAGCTATGCTGTTTTGAATGGT CCCAAAACAACATTGCCGATGATAACTTGAG

AAAGAGGGTTAATACCAGCAGTCGGATACCTTCCTATTCTTTCTGTTAAAGCGTTTT CATGTTATAATAGGCAAAAGAAGAGTAGTGT GATGGAACAAACATTTTTTATGATTAAGCCATATGGGGTTAAGCAAGGGGAGGTAGTTGG AGAGGTTTTACGGTGGATTGAACGCCTA AGATTTACGTTTAAGCGATTCGAGCTAAGACAAGCTAGTTCGAAATACTTGGCTAAGCAC GACGAGGCCTTGGTGATAAACCTTTTGA TCCTAAACTTAAAGCTTACATGACAAGTGGTCCTGTTTTAATTGGGATAATTCTTGGGGA CTAAGGTGGTATCGTCCATTCCGACAGC ATCGCCAGTCAC

PCR conditions to generate the three fragments were:

Initial Denaturation 98°C_60 sec 98°C_60 sec 98°C_60 sec

35 Cycles 98°C_10 sec 98°C_10 sec 98°C_10 sec

64°C_30 sec 62°C_30 sec 62°C_30 sec

72°C_240 sec 72°C_30 sec 72°C_30 sec

Final Extension 72°C_120 sec 72°C_120 sec 72°C_120 sec

Hold 4 °C 4 °C 4 °C

Results showing the PCR amplicons are provided in Figure 1 1 .

Assembly of pNB100 from three PCR amplicons, tracrRNA-cas9, leader and first direct repeat, second direct repeat; plus pACYC184 digested with EcoRV

We employed a Gibson assembly kit from NEB (E5510) and followed the protocol provided by the manufacturer to assemble the above three PCR amplicons along with pACYC184. The component of each fragment in the assembling reaction is shown in the following table.

2 μΙ_ of the assembly reaction was transformed to DH5a competent cells (purchased from New England Biolabs) followed by selection on chloramphenicol (35pg/ml_) LB plates. The recombinants were screened by PCR using the three primer sets used for obtaining the initial three fragments. The plasmid templates giving three desired amplicons were isolated from the candidate clones and were subjected to sequence analysis.

The sequence of the final construct of pNB100 (SEQ ID NO: 145)

GAATTCCGGA TGAGCA TTCA TCAGGCGGGCAAGAA TGTGAA TAAAGGCCGGA TAAAACTTGTGCTTA TTTTTCTTTACGG TCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTG ACTGAAATGCCTCAAAATGT

49

SUBSTITUTE SHEET RULE 26 TCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATT TTAGCTTCCTTAGCTCCTGA AAA TCTCGA TAACTCAAAAAA TACGCCCGGTAGTGA TCTTA TTTCA TTA TGGTGAAAGTTGGAACCTCTTACGTGCCGA T CAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAG GATTTATTTATTCTGCGAAG TGA TCTTCCGTCACAGGTA TTTA TTCGGCGCAAAGTGCGTCGGGTGA TGCTGCCAACTTACTGA TTTAGTGTA TGA TGGT GTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTG ACGGGGTGGTGCGTAACGGC AAAAGCACCGCCGGACATCAGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTG ATGAGGGTGTCAGTGAAGTG CTTCA TGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAA TA TGTGA TACAGGA TA TA TTCCGCTTCCTCGCTC ACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGC GGAGATTTCCTGGAAGATGC CAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTC CGCCCCCCTGACAAGCATCA CGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC GTTTCCCCCTGGCGGCTCCC TCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGC GTTTGTCTCATTCCACGCCT GACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGT TCAGTCCGACCGCTGCGCCT TATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAG CAGCCACTGGTAATTGATTT AGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGG TGACTGCGCTCCTCCAAGCC AGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGG CGGTTTTTTCGTTTTCAGAG CAAGAGA TTACGCGCAGACCAAAACGA TCTCAAGAAGA TCA TCTTA TTAA TCAGA TAAAA TA TTTCTAGATTTCAGTGCA ATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTGTAATTCTC ATGTTTGACAGCTTATCATC GA TAAGCTTTAA TGCGGTAGTTTA TCACAGTTAAA TTGCTAACGCAGTCAGGCACCGTGTA TGAAA TCTAACAA TGCGCT CATCGTCATCCTCGGCACCGTCACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGT ACTGCCGGGCCTCTTGCGGG ATCCAGAAGTCTTTTTCTTGCACTGTTTCCTTTTCTTTATGATAGTTTACGAAATCATCC TGTGGAGCTTAGTAGGTTTA G C AAGAT GGCAGCGCC T AAAT G T AGAAT GAT AAAAG GAT T AAGAGAT T AAT T T C C C T AAAAAT GAT AAAAC AAG C G T T T T GAAAGCGCTTGTTTTTTTGGTTTGCAGTCAGAGTAGAATAGAAGTATCAAAAAAAGCACC GACTCGGTGCCACTTTTTCA AGTTGATAACGGACTAGCCTTATTTTAACTTGCTATGCTGTTTTGAATGGTTCCAACAAG ATTATTTTArAACrrrrArA ACAAATAATCAAGGAGAAATTCAAAGAAATTTATCAGCCATAAAACAAT ΑΟΎΎΑΑΎ ACT ATAGAATGATAACAAAAT AAA CTACTTTTTAAAAGAATTTTGTGTTATAATCTATTTATTATTAAGTATTGGGTAATATTT TTTGAAGAGATATTTTGAAA AAGAAAAAT T AAAG CAT AT T AAAC T AAT T T C G GAG G T C AT T AAAAC TAT TAT T GAAAT CAT C AAAC TCATTATG GAT T T A

GG A ACACGT^CGGAAGAATCG A ^T ^G ^TATCTACAG AGA ^T ^T ^TCAAATGAGATGGCGAAAGTAGATGATAGTTTC TTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCT ATTTTTGGAAATATAGTAGA T GAAG TTGCTTATCAT GAGAAAT AT C C AAC TATCTATCATCTGC GAAAAAAAT T G G T AGAT T C T AC T GAT AAAG C G GAT T TGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTG AGGGAGATTTAAATCCTGAT AAT AG T GAT G T G GAC AAAC TATTTATCCAGTTGG T AC AAAC C T AC AAT C AAT T AT T T GAAGAAAAC C C T AT TAAC G C AAG TGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAA TCTCATTGCTCAGCTCCCCG GTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTA ATTTTAAATCAAATTTTGAT T T G G C AGAAGAT G C T AAAT TACAGCTTT C AAAAGAT AC T T AC GAT GAT GAT T T AGAT AAT TTATTGGCG C AAAT T G GAGA TCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGA TATCCTAAGAGTAAATACTG AAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAG ACTTGACTCTTTTAAAAGCT T T AG T T C GAC AAC AAC T T C C AGAAAAG T AT AAAGAAAT C T T T T T T GAT C AAT C AAAAAAC G GAT AT GCAGGTTATATT GA T G G G G GAG C T AG C C AAGAAGAAT T T TAT AAAT T T AT C AAAC C AAT T T T AGAAAAAAT G GAT G G T AC T GAG GAAT T AT T G G TGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTC CCCATCAAATTCACTTGGGT GAGCTGCATGCTATTTT GAGAAGAC AAGAAGAC TTTTATCCATTTT T AAAAGAC AAT C G T GAGAAGAT T GAAAAAAT C T T GACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATG GATGACTCGGAAGTCTGAAG AAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCAT TTATTGAACGCATGACAAAC T T T GAT AAAAAT C T T C C AAAT GAAAAAG T AC T AC C AAAAC AT AG TTTGCTTTAT GAG TATTTTACGGTT TAT AAC GAAT T GAC AAAG G T C AAAT AT G T T AC T GAAG GAAT G C GAAAAC CAGCATTTCTTTCAGGT GAAC AGAAGAAAG C CAT T G T T GAT T T AC T C T T C AAAAC AAAT C GAAAAG TAAC C G T T AAG C AAT T AAAAGAAGAT T AT T T CAAAAAAAT AGAAT G T T T T GAT AG T G T T GAAAT T T C AG GAG T T GAAGAT AGAT T T AAT GCTTCATTAGGTACCTACCAT GAT T T G C T AAAAAT TAT T AAAGAT AA AGAT T T T T T G GAT AAT GAAGAAAAT GAAGAT AT C T T AGAG GAT AT T G T T T TAAC AT T GAC C T T AT T T GAAGAT AG G GAGA T GAT T GAG GAAAGAC T T AAAAC AT AT GCTCACCTCTTT GAT GAT AAG G T GAT GAAAC AG C T T AAAC GTCGCCGTTATACT GGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAA ACAATATTAGATTTTTTGAA AT C AGAT GGTTTTGC C AAT C G C AAT TTTATGCAGCT GAT C C AT GAT GAT AG T T T GAC AT T T AAAGAAGAC AT T C AAAAAG C AC AAG T G T C T G GAC AAG G C GAT AG T T T AC AT GAAC AT AT T G C AAAT TTAGCTGGTAGCCCTGCTAT T AAAAAAG G T AT T TTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAA AATATCGTTATTGAAATGGC AC G T GAAAAT C AGAC AAC T C AAAAG G G C C AGAAAAAT T C G C GAGAG C G T AT GAAAC GAAT C GAAGAAG G T AT CAAAGAAT TAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGC TCTATCTCTATTATCTCCAA AAT G GAAGAGAC AT G T AT G T G GAC C AAGAAT T AGAT AT T AAT C G T T T AAG T GAT TAT GAT G T C GAT CAC AT TGTTCCACA AAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCG TGGTAAATCGGATAACGTTC C AAG T GAAGAAG T AG T C AAAAAGAT GAAAAAC T AT T G GAGAC AAC T T C T AAAC G C C AAG T T AAT C AC T C AAC G T AAG T T T GATAAT T TAAC GAAAGC T GAAC GTGGAGGTTTGAGT GAAC TTGATAAAGCTGGTTT TAT CAAACGCCAATTGGTT GAAAC T C G C C AAAT CAC T AAG C AT G T G G CAC AAAT T T T G GAT AG T C G C AT GAAT AC T AAAT AC GAT GAAAAT GAT AAAC Ύ ΤΑΤΤΟ GAGAGGrrAAAGrGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCC AATTCTATAAAGTACGTGAG

50

SUBSTITUTE SHEET RULE 26 ATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTG ATTAAGAAATATCCAAAACT TGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAA GTCTGAGCAAGAAATAGGCA AAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAAT ACACTTGCAAATGGAGAGA CGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGG CGAGATTTTGCCACAGTGCG CAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGG ATTCTCCAAGGAGi'CAA'I'T ⁷ ! ¹ ACCAftAfiAGAfiAT CGGACAAGC TAT GCTCG AAftAfiAG

AC^GTAGCT A TCAGTCC AGTGG TGC AAGGTGGAAAAAGGGAAATCG

AGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTT^^

AAAAAGACTTAATCATTAAACTACCTAAAT T GTCTTTTTGAGTTA^^

GGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTΊΑ� �ATTΊAGCΊAGTCATTATGAAAA GTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCA TTATTTAGATGAGAT'TATT'G AGCAAATCAGTGAATΐTΐCTAAGCGTGTTATΐTTAGCAGATGCCAATΐTAGATAAA GTTCTTAGTGCATATAACAAA"AT ¹ AGAGACAAACCAATACGTGAACAAGCAGAAAARAT ^: TATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTT TAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGA TGCCACTCTTATCCATCAAT CCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGATGGC CACGTGAACTATATGATTTT CCGCAGTATATTTTAGATGAAGATTATTTCTTAATAACTAAAAATATGGTATAATACTCT ^AATAAATGCAGTAATACAG GGGCTTTTCAAGA TGAAGTCTAGCTGAGACAAATAGTGCGATTACGAAATTTTTTAGACAAAAATAGTCTACG AGGTTT TAGAGCTATGCTATTTTGAATGGTCCCAAAACTGAGACCAGTCTCGGACGRCCAAAGGRC RCGTTTTAGAGCTATGCTGT

TTTGAATGGTCCCAAAACAACATTGCCGATGATAACTTGAGAAAGAGGGTTAATACC AGCAGTCGGATACCTTCCTATTC TTTCTGTTAAAGCGTTTTCATGTTATAATAGGCAAAAGAAGAGTAGTGTGATGGAACATA CATTTTTTATGATTAAGCCA TATGGGGTTAAGCAAGGGGAGGTAGTTGGAGAGGTTTTACGGTGGATTGAACGCCTAAGA TTTACGTTTAAGCGATTCGA GCTAAGACAAGCTAGTTCGAAATACTTGGCTAAGCACGACGAGGCCTTGGTGATAAACCT TTTGATCCTAAACTTAAAGC TTACATGACAAGTGGTCCTGTTTTAATTGGGATAATTCTTGGGGACTAAGGTGGTArCGr CCArrCCGACAGCArCGCCA GTCACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTC TCGGAGCACTGTCCGACCGC TTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGAGCCACTATCGACTACGCGATC ATGGCGACCACACCCGTCCT GTGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGC TGGCGCCTATATCGCCGACA TCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGG GTATGGTGGCAGGCCCCGTG GCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTC AACGGCCTCAACCTACTACT GGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCCCTTGAGAGC CTTCAACCCAGTCAGCTCCT TCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGC AACTCGTAGGACAGGTGCCG GCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGC CTGTCGCTTGCGGTATTCGG AATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGA GAAGCAGGCCATTATCGCCG GCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGG CCTTCCCCATTATGATTCTT CTCGCTTCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGAT GACGACCATCAGGGACAGCT TCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCATTGGACCGCTGATCGTCAC GGCGATTTATGCCGCCTCGG CGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCC CCGCGTTGCGTCGCGGTGCA TGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCA CTCCAAGAATTGGAGCCAAT CAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCG TCCGCCATCTCCAGCAGCCG CACGCGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCT GTCGTTGAGGACCCGGCTAG GCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAA GCGACTGCTGCTGCAAAACG TCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAA ACGCGGAAGTCCCCTACGTG CTGCTGAAGTTGCCCGCAACAGAGAGTGGAACCAACCGGTGATACCACGATACTATGACT GAGAGTCAACGCCATGAGCG GCCTCATTTCTTATTCTGAGTTACAACAGTCCGCACCGCTGTCCGGTAGCTCCTTCCGGT GGGCGCGGGGCATGACTATC GTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCG CCCAACAGTCCCCCGGCCAC GGGGCCTGCCACCATACCCACGCCGAAACAAGCGCCCTGCACCATTATGTTCCGGATCTG CATCGCAGGATGCTGCTGGC TACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTAACCGTTTTTATCAGGCTCTG GGAGGCAGAATAAATGATCA TATCGTCAATTATTACCTCCACGGGGAGAGCCTGAGCAAACTGGCCTCAGGCATTTGAGA AGCACACGGTCACACTGCTT CCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGC CCTGAACCGACGACCGGGTC GAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCAC CAGGCGTTTAAGGGCACCAA TAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCA TTAAGCATTCTGCCGACATG GAAGCCATCACAGACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCC TTGCGTATAATATTTGCCCA TGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGA AACTCACCCAGGGATTGGCT GAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAA CACGCCACATCTTGCGAATA TATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTC AGTTTGCTCATGGAAAACGG TGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACG

The total number of nucleotides is 9578 bp. The backbone vector pACYC184 sequence is italicised, sequence positions are numbered from G of EcoRI site (GAATTC) underlined. tracrRNA is located at nucleotide No. from 1889 to 1974 indicated bold letters, Cas9 D initiation and termination codons are indicated by bold three letters, starting at

51

SUBSTITUTE SHEET RULE 26 nucleotide No. 2270 and ending at 6376 followed by leader sequence 6462-6556 indicated by italicised bold letters, first and second direct repeat sequences are underlined, between which spacer cloning region (30 mer) is located. This spacer cloning region contains two inverted Bsa\ sites indicated by bold italicised letters 5'-GAGACC-3' and 5'-GGTCTC-3' for creating 5' four bases protruding spacer cloning sites 5'-GTTT-3' and 5'-TTTT-3', respectively and one unique AatW (5'-GACGTC-3') site also indicated by bold italicised to reduce self-ligation in the event of incomplete Bsa\ digestion. Note the transition and transversion base changes G6573A, A6779T that were detected by Sanger sequencing and are shown in bold letters, respectively. However, these point mutations do not affect the CRISPR-Cas9 activity, which will be shown in the later section. The two unique sites, Sal I (GTCGAC) and Xba I (TCTAGA) highlighted in bold italicised letters are utilised to isolate the CRISPR/Cas9 construct for cloning into M13mp18.

A plasmid map of pNB100 is shown in Figure 12.

The desired spacer sequence can be cloned in the clockwise direction between Bsa\ sites. This vector contains the p15A origin at 1393-848 and cat (chloramphenicol resistant) gene at 219-9137. Cutting positions of each restriction enzyme, indicated in the parentheses, refer to the position of the 5' cutting sites on the top strand within the recognition sequence.

Construction of pNB102

pNB100 was digested with Bsa\ and AatW followed by purification using Agencourt ampure beads. The spacer sequence CR30 was employed from the discussion in B. Selection of spacer sequence from the target sequence in the Materials and Methods section above. The CR30 sequence is as follows :

5 ' -AAACACTTTAAAAGTGCTCATCATg (SEQ ID NO: 117)

tgaaattttcacgagtagtacAAAA-5' (SEQ ID NO: 118)

This double-stranded DNA cassette is generated by denaturation at 95 C for 1 min and re- annealing at -1 degree every min to 30 C in the 1 x T4 ligase buffer (50 mM Tris-HCI(pH 7.5 at 25 C), 10 mM MgCI2, 1 mM ATP 10 mM DTT) plus 50 mM NaCI following the kinase reaction to add a phosphate moiety in the 5' terminus of each oligo. The annealed cassette contains 5' protruding four base compatible bases on both ends for the sites created on pNB102 by Bsa\ digestion. This CR30 cassette was ligated to pNB100 by T4 DNA ligase and transformed to DH5a competent cells (purchased from New England Biolabs). The

52

SUBSTITUTE SHEET RULE 26 transformants were selected on chloramphenicol LB plate and were screened by PCR with the bottom sequence of the CR30 cassette as a reverse primer and a forward primer CF1 : 5'-acgttgacgaatcttggagc, which anneals at 6209-6228 region on the recombinant plasmid to generate 409 bp PCR amplicon. PCR positive clones were sequenced to confirm the CR30 spacer sequence and this recombinant clone is designated as pNB102 and used for in vitro beta lactamase-gene disruption experiments. CR30 spacer anneals to the sense strand of beta lactamase-gene and cleaves the phosphodiester bonds between 188th and 189th nucleotide on the sense and antisense strand.

Construction of M13mp18::NB102

pNB102 was digested with unique restriction sites Sa/I and Xba\ to generate two fragments 6044 bp and 3524 bp. The fragment length was calculated from the 5' end of the restricted sites in the top strand within the restriction recognition sites. The 6044 bp fragments containing CRISPR locus with CR30 spacer sequence in the CRISPR array was separated from the 3524 bp fragment and purified on the preparative 1 % agarose gel. M13 mp18 was digested with Sal\-Xba\, followed by Agencourt ampure purification to remove the six bases Sal\-Xba\ fragment from the reaction. These two purified fragments were combined and ligated by T4 DNA ligase and transformed to DH5aF'lq competent cells (purchased from New England Biolabs). Transformed cells were plated along with freshly grown DH5aF'lq cells as a phage indicator and IPTG/X-gal solution as a blue-white colour indicator with molten top agar to LB plate. White plaques collected from the lawn were screened by PCR for the presence of the CR30 spacer sequence. The correct phage constructs obtained were purified by two times single plaque isolation. The entire sequence length of the final construct is 13288 bp. This spacer CR30 positive recombinant is designated as M13mp18::NB102 and was used for the bla-gene inactivation experiments mediated by M13 phage delivery.

Delivery of CRISPR-Cas9 constructs to bacteria

Having constructed the CRISPR-Cas9 plasmid pNB100 and the derivative plasmid pNB102 carrying a spacer insertion targeted against the beta-lactamase (bla) genes encoded by the bacterial transposable elements Tn1 and Tn3, we then sought to demonstrate, using three delivery methods, (i) plasmid conjugation, (ii) plasmid DNA transformation, (iii)

bacteriophage infection, that bacterial cells carrying copies of the CRISPR-Cas9 construct with bla-spacer insertion would be unable to grow in the presence of the beta-lactam antibiotic ampicillin. Constructs that are able to inactivate target genes, including antibiotic resistant genes, via the CRISPR-Cas system, and which are an aspect of the present invention are also referred to herein as "Nemesis symbiotics".

5

"Nemesis symbiotic activity" (NSA) assay by plasmid conjugation: a prophylaxis experiment

We demonstrate here that Nemesis symbiotics can prevent the spread of antibiotic resistance by inhibiting conjugal transfer of conjugative plasmids carrying antibiotic

10 resistance genes from a donor cell to a recipient cell carrying the Nemesis symbiotics. To do this we mated a recipient cell carrying the Nemesis symbiotics with a donor cell carrying a conjugative plasmid, plus a multicopy mobilisable plasmid carrying the bla gene encoding ampicillin resistance. In a successful mating, the conjugative plasmid will transfer itself plus the mobilisable plasmid carrying ampicillin resistance to the recipient. Exconjugants may be

15 selected for resistance to both chloramphenicol present on a non-mobilisable plasmid in the recipient and ampicillin received from the donor. Successful Nemesis symbiotic activity will reduce the efficiency of transfer of ampicillin resistance.

The recipient cell DH5a (F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG

20 08OdlacZAM15 A(lacZYA-argF)U169, hsdR17(rK- mK+), λ-) was purchased from New England Biolabs and transformed with the plasmids pNB100 or pNB102 or pACYC184, where plasmids encode chloramphenicol resistance and both pNB100 and pNB102 carry CRISPR-Cas9 but only pNB102 carries the spacer sequence targeted against the beta- lactamase gene. The plasmid pACYC184 is the non-mobilisable parent plasmid used for 25 the construction of pNB100 and pNB102 as described above.

The donor strain JA200 (F+ thr-1 , leu-6, DE(trpE)5, recA, lacY, thi, gal, xyl, ara, mtl) also carrying plasmid pNT3 is described by Saka et al. DNA Research 12, 63-68 (2005). The plasmid pNT3 is a mobilisable plasmid carrying the bla gene of Tn1 .

A single colony of the donor JA200 pNT3 was picked from a Luria broth (LB) plate

30 containing 100 μg/mL Ampicillin and grown shaking at 37°C overnight in 1 mL LB medium with 100 μg/mL Ampicillin. A single colony each of the recipients, DH5a pNB100 and DH5a pNB102 was picked from a LB plate containing 35 μg/mL Chloramphenicol and grown shaking at 37°C overnight in 1 mL LB with 35 μg/mL Chloramphenicol. To wash cells to remove antibiotics, 50μί of cells were added to 1 mL LB in Eppendorf tubes and centrifuged 60 sec at 12500 rpm. Cells were resuspended in 50μΙ_ LB. To set up the mating JA200 pNT3 was spotted onto an LB plate, then 2μΙ of each DH5a carrying pNB100 and pNB102 were added to this spot. Separate 2μΙ spottings of donor and recipients were also performed (i.e. not mated). Plates were incubated at 37 °C for 4 hours. Cells were removed resuspended in LB and 100μί plated on LB plates containing both 100 μg/mL Ampicillin and 35 μg/mL Chloramphenicol (LB ApCm). 10Ομί of 10,000 fold (10 ^Λ -4) dilutions were also plated on LB plates and incubated at 37 °C overnight. The resultant colonies were counted as shown in the Table below.

Photographs in Figure 13 show platings of the matings between: (A) JA200 pNT3 x DH5a pNB100 (as expected lacking Nemesis symbiotic activity); and (B) JA200 pNT3 x DH5a pNB102 (showing Nemesis symbiotic activity).

The 10 ^Λ -4 dilution plated on LB plates, gave approximately 500 colonies a count of 5 x 10 ^Λ 7 cells per mL in the mated cell suspension and for the JA200 pNT3 x DH5a pNB102 only 3.7 x 10 ^Λ 2 cells/mL were able to grown on the LB Ap100Cm35 plates. Thus assuming half the cells are recipients/ex-conjugants then: 3.7 x 10 ^Λ 2 cells /ml divided by 2.5 x 10 ^Λ 7 gives a mating efficiency for the recipient carrying pNB102 of 1 .2 x 5 x 10 ^Λ -5 The experiment demonstrated a significant reduction in CmRApR exconjugants in matings with DH5a carrying pNB102 versus pNB100 x JA200 pNT3. In order to measure the relative mating efficiencies more accurately, after a liquid mating cells were plated on LB plates to titre all cells, LB Ap1 00 plates to titre donors plus exconjugants, LB Cm plates to titre recipients plus exconjugants and LB Ap100Cm35 to titre exconjugants only. For the liquid mating overnight cultures of 10μί of JA200 pNT3 were mixed with 10μί of recipients DH5a pNB100 or DH5a pNB1 02 200μί of LB added and tubes incubated overnight at 37 ^< €. Mating mixtures were diluted 10 ^Λ -1 , 10 ^Λ -3, 10 ^Λ -5 in LB and 50μΙ of dilutions plated on LB, LB Ap100Cm35, LB Ap100 and LB Cm35 plates and plates incubated overnight at 37 °C. The table below summarises the cell titres obtained.

The number of cells on LB Cm plus LB Ap plates should equal the number of cells on LB plates. For pNB100 1 .40 x 10 ^Λ 8 (Cm plates) plus 2.64 x 10 ^Λ 8 (Ap plates) = 4.04 x 1 0 ^Λ 8 which agrees very well with 4.14 x 1 0 ^Λ 8 on LB plates. For pNB102 1 .78 x 1 0 ^Λ 8 (Cm plates) plus 3.82 1 0 ^Λ 8 (Ap plates) = 5.6 x 10 ^Λ 8 which agrees very well with 5.16 x 10 ^Λ 8 on LB plates.

In conclusion, the data show that after overnight mating in liquid culture, there is a 5,000 fold reduction in mating efficiency per recipient comparing pNB102 with the spacer to pNB100 lacking the spacer. NSA Assay by plasmid transformation

In this experiment, we demonstrate that introduction of Nemesis symbiotics to recipient cells by DNA transformation inactivates antibiotic resistance in the transformants. In order to obtain a tester strain, DH5a competent cells purchased from New England Biolabs were transformed with pBR322 (carrying the bla gene derived from Tn3) and selected on LB Ap100 plates. Competent cells of the derived strain DH5a pBR322 were then prepared using the CaCI2 protocol 25 (1 .1 16) as described by Sambrook and Russell in Molecular Cloning: A Laboratory Manual (3rd Edition, 2001 ) and subsequently transformed with plasmids pNB100, pNB102 and pACYCI 84 with selection for CmR. Transformant colonies were then picked onto LB Cm35 and LB Ap100 plates. Primary transformants were replica toothpicked onto both LB Cm35 and LB Ap100 plates and incubated overnight at 37 °C. The results, depicted in Figure 14, show that all colonies toothpicked from DH5a pBR322 transformed by pNB100 (lacking the bla gene target spacer sequence) remain resistant to ampicillin. In contrast all colonies toothpicked from DH5a pBR322 transformed by pNB102 have lost ampicillin resistance, so demonstrating Nemesis symbiotic activity. The experiments above do not give a value for the fraction of primary transformants where NSA has inactivated the bla gene. To address this, single colonies from the primary transformants were picked into 1 mL LB and diluted 10 ^Λ -3 in LB. Then 100μί plated onto plates as follows, and results scored. The results showed that following transformation of DH5a pBR322 with pNB102 fewer than 10 ^Λ -6 cells retain ApR. Nemesis symbiotic activity is very efficient.

NSA Assay by bacteriophage M13 infection

In this experiment, we demonstrate that introduction of Nemesis symbiotics to recipient cells by bacteriophage infection inactivate antibiotic resistance in the transformants. We chose the male-specific filamentous phage M13 as the delivery agent for the Nemesis symbiotic construct. An M13 derivative M13mp18::NB102 carrying and the Cas9 CRISPR plus bla gene target spacer region of pNB102 was used to deliver the Nemesis symbiotic by infection of an F+ strain, JA200, carrying ampicillin resistance on the plasmid pNT3. 0.2mL of a fresh culture of this strain was added to 3 mL of LB top agar (Luria broth with 0.7% agar) and poured onto an LB plate. Then 2μΙ of phage stocks of M13mp18::NB102 (10 ^Λ 8 pfu/mL) and as a control M13mp18 were spotted onto the lawn and the plate was incubated 8 hours at 37°C. Plaques were picked into 1 .5 mL LB and grown shaking o/n at 37 °C. A control strain DH5a lacking ampicillin resistance was also cultured overnight from a single colony picked into 1 .5 mL LB. Nitrocefin assay for beta lactamase activity was performed:

1 mL of the culture of cells was centrifuged for 60 sec at 12,500 rpm in microfuge then 2μΙ of stock nitrocefin (10mg/mL in DMSO) was added to 1 mL of cell supernatant and absorbance of the degradation product of nitrocefin was measured at 482 nm in a spectrophotometer several time points after addition of nitrocefin. The Table below summarises the results.

The experiments reported above provide the proof-of-concept that, in the model organism, Escherichia coli, DNA constructs carrying the Cas9 CRISPR region plus a spacer region with sequences directed against a target region of the beta-lactamase gene can inactivate ampicillin resistance when delivered by naked DNA transformation and bacteriophage infection as well as prevent transfer of ampicillin resistance by plasmid conjugation. It is apparent that Nemesis symbiotics of the invention can be applied to pathogenic bacteria and for other antibiotic resistance genes.

Example 3

The aim of Exampie 3 is to extend the proof-of-concept for resurrection of antibiotic efficacy by introduction of a CRISPR/Cas9 construct in a pathogenic bacterial strain of Klebsiella pneumoniae. Resistance to a newer class of beta-lactam antibiotics, the Carbapenems has emerged in Enterobacteriaceae by the acquisition of new bla genes that encode beta- lactamases able to degrade even the Carbpenems. Klebsiella pneumoniae strains with resistance to Carbapenems, KPC are important causes of morbidity and mortality among hospital-acquired and long-term care-associated infections. Noteworthy is the very high mortality (around 30-70%) among patients with bacteraemia or pulmonary infections. Example 3 shows thai a Klebsiella pneumoniae carrying a beta- lactamase (bla) gene conferring resistance to the beta-lactam antibiotic, ampiciliin become sensitive to ampiciiliri following introduction of a modified CRISPR/Cas9 construct targeted against the bla gene. The same CRISPR/Cas9/anti-i?/a construct used in Example 1 or Example 2 is used and is delivered to Klebsiella pneumoniae by conjugation with a donor strain carrying a conjugative plasmid with CRISPR/CasS/anti-d/a. Ex-conjugant recipient Klebsiella pneumoniae carrying the plasmid with CRiSPR/Cas9/anti-6/a construct is selected for on appropriate media with counter-selection against the donor cell and Klebsiella pneumoniae cells that failed to receive the plasmid.

As for Example 1 and Example 2, efficacy of the CRISPR/Cas9/anti-£)/a contstruct is evaluated by comparison to a negative control of a Klebsiella pneumoniae ex-conjugant strain that received a plasmid with CRISPR/Cas9 (i.e. lacking the anti-o/a region). As in Example 1 and Example 2, the beta-laciamase activity can be detected by nitrocefin to count white versus red colonies. Also as in Example 1 and Example 2, alternatively beta lactamase activity is seen directly challenging the bacteria on the LB plate with/without ampiciliin and measuring the fraction of the ampiciliin sensitive colonies versus ampiciliin resistant ones. The identical experiment to that described above is also evaluated using instead a

CRISPR/Cas9/anti-d/aCfc, where the CRISPR/Cas9/anti-i)/aCi> construct carries a region, anti-d/aCo, targeted against a bla gene encoding a beta-lactamase able to break down carbapenems and present in that particular Klebsiella pneumoniae strain.

Example 4

Example 4 provides delivery routes for the therapeutic constructs. These routes all apply to veterinary as well as human applications to be delivered orally, topically, probiotically and for use in surgical irrigation fluids and wound dressings. Orally: Phage containing assassin construct as the active ingredient may be administered orally as a stabilised therapeutic preparation: either - administered before antibiotic therapy or administered in the form of an adjuvant complexed to an antibiotic. Conjugative plasmids containing assassin construct as the active ingredient may be administered as a culture of commensal bacteria carrying these plasmids in order to transmit these plasmids to gut flora thereby generating prophylactic protection against future infection with antibiotic resistant bacterial pathogens. Topically: Phage containing assassin construct as the active ingredient may be administered topically as a stabilised medication (ointment, spray, powder etc): either - administered before antibiotic topical medication; or administered in the form of a complex with an antibiotic medication. Topical application of conjugative plasmids containing assassin construct as the active ingredient may be via a stabilised culture of commensal bacteria carrying these plasmids in order to transmit the plasmids to gut flora thereby generating prophylactic protection against future infection with antibiotic resistant bacterial pathogens. Probiotically: Phage containing assassin construct as the active ingredient may be administered probiotically as a stabilised culture of commensal bacteria (e.g. Lactobacillus spp) carrying these plasmids in order to transmit the plasmids to gut flora thereby generating prophylactic protection against future infection with antibiotic resistant bacterial pathogens. One example of this may be the probiotic, prophylactic administration of such a preparation to patients in settings with a high risk of infection with antibiotic resistant bacterial pathogens such as hospitals, care homes, schools, transplantation centres etc. Another example may be the administration of such a preparation to livestock via animal feeds, thereby limiting the rise and horizontal transmission of antibiotic resistant bacteria. The use of the present invention in livestock thus represents one means for (indirect) prophylactic treatment of antibiotic resistant bacteria in humans.

Surgical irrigation fluids: Phage containing assassin construct as the active ingredient may be added to surgical irrigation fluids and also sprays for disinfecting fomites. Stabilised commensal bacterial cultures containing conjugative plasmids incorporating assassin constructs may be added as coatings to surgical wipes and to wound dressings.

Example 5

In this example, a construct is designed to include multiple RNA guide molecules where each RNA guide molecule is transcribed by its own promoter.

Figure 16 exemplifies a set of spacer sequences that we have identified encoding 20 guide RNA molecules targeted against 1 17 different bla genes identified in the NCBI ARDB database for Klebsiella pneumoniae. The beta lactamase gene type, spacer sequence and antibiotic resistance profile in Klebsiella pneumoniae obtained from NCBI ARDB database are shown. Beta lactamase gene sequences were collected from the ARDB database with the keyword Klebsiella pneumoniae. Redundant sequences were removed and unique sequences used for multiple sequence alignment using web program Clustal Omega. One canonical sequence was chosen from each cluster and the 20 nt spacer sequences predicted by the web program Jack Lin's CRISPR/Cas9 gRNA finder collected. The spacer sequence was chosen to maximise the ratio of the proto-spacer sequence found in the sequences belonging to the same branch. Each of the example spacer sequences shown in the 4 ^th column has the capability to disrupt the genes in the third column. Beta lactamase genes used in this analysis are: SHV-a = 1 , 2, 2a, 5, 5a, 1 1 , 12, 14, 26, 27, 28, 31 , 33, 38, 43, 44, 48, 55, 56, 60, 61 , 62, 71 , 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82,85, 89, 92, 98, 99, 101 , 103, 106, 107, 108, 109, CTXM-b = 1 , 3, 10, 12, 15, 22, 32, 54, 60, 62, 68, CTXM-c = 13, 14, 16, 18, 19, 24, 26, CTXM-d = 2, 35, 59, CTXM-e = 26, 63, TEM-f = 1 , 1 b, 3, ESBL, 139, KPC-g = 1 , 2, 3, 4, OKP-h = A1 1 , A12, A16, A17, B13, B-SB613, 6, LEN- i = 2, 17, 18, 19, 20, 21 , 22, 24,GES-j = 1 , 3, 4, 5, VIM-a = 1 , 2, 4, 12, 19, IMP-b = 4, 8, CMY-a = 2, 4, 25, 31 , 36, LAT-b = 1 , 2, CMY-c = 1 , 8b, 10, 19, FOX-d = 1 , 5, 7, OXA-a = 1 , 30, 47, OXA-2, OXA-9, OXA-b = 10, 17.

Beta lactam antibiotics were classified into four classes, penams, cephems, carbapenem and monobactam. One antibiotic name is listed in Figure 16 as an example under each class. The beta lactamase, which can open the beta lactam ring is indicated by R. For example, carbapenem is inactivated by KPC. To re-sensitise bacteria to carbapenem, the spacer sequence 5'-TTGTTGCTGAAGGAGTTGGG (SEQ ID NO: 45) should be employed into the spacer array and inactivate KPC genes. Note that the spacer sequence for CMY-a can be employed to LAT-b cleavage.

Similarly, Figure 17 exemplifies a set of spacer sequences encoding 17 guide RNA molecules targeted against 154 different bla genes identified in the CARD database for Klebsiella pneumoniae. Beta lactamase gene type, spacer sequence and antibiotic resistance profile in Klebsiella pneumoniae obtained from the IIDR CARD database are shown.

Beta lactamase gene sequences were collected by filtering all the collected beta lactamase genes with the keyword Klebsiella pneumoniae and subjected to multiple sequence alignment using web program Clustal Omega. One canonical sequence from each cluster was chosen and the 20 nt spacer sequences predicted by the web program Jack Lin's CRISPR/Cas9 gRNA finder collected. The spacer sequence was chosen to maximise the ratio of the proto-spacer sequence found in the sequences belonging to the same branch. The each of the example spacer sequences shown in the 4 ^th raw has the capability to disrupt the genes in the third column. Beta lactamase genes used in this analysis are: SHV-a = 1 a, 5, 2a,1 1 , 18, 20, 21 , 22, 23, 28, 31 , 32, 52, 55, 98, 99, 100, 106, 107, 1 10, 1 1 1 , 121 , 134, 136, 137, 140, 143, 144, 147, 148, 149,150, 151 , 152, 153, 154, 155, 157, 158, 159, 160, 161 , 162,163, 164, 165, 168, 172, 173, 178,179, CTXM-b = 10, 12, 15, 52, 54, 62, 71 , 80, CTXM-c = 19, 81 , 99, 147, TEM-f = 1 , 162, 183, 192, 197, 198, 209, KPC-g = 3, 4, 6, 7, 8, 1 1 , 12, 14, 15, 16, 17, OKP- h = 5, 6, A1 , A2, A4, A5, A6, A7, A8, A9 A10, A1 1 , A12, A13, A14, A15, A16, B1 , B2, B3, B4, B5, B6, B7, B8, B9, B10, B1 1 , B12, B13, B17, B18, B19, B20, LEN-i = 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 18, 19, 20, 21 , 22, 23, 24, GES-j = 1 , 3, 4, VIM-a = 4, 26, 27, 28, 33, 34, IMP-b = 32, 38, NDM-c = 1 , 9, 10, ACT-3, CMY-a = 25, 31 , 56, 87, FOX-d = 8, 9, OXA-a = 1 , 30, 47, OXA-1 , OXA-a = 181 , 204, 247, OXA-9.

Beta lactam antibiotics are classified into four classes, penams, cephems, carbapenem and monobactam. In Figure 17, one antibiotic name is listed as an example under each class. The beta lactamase, which can open the beta lactam ring is indicated by R. For example, carbapenem is inactivated by KPC. To re-sensitize bacteria to carbapenem, the spacer sequence 5'-TTGTTGCTGAAGGAGTTGGG (SEQ ID NO: 45) should be employed into the spacer array and inactivate KPC genes.

Example 6

In this example, a modified Cas DNA-binding polypeptide is created that deletes and reseals rather than leaving a DSB.

As described above, DSBs caused by Cas DNA-binding polypeptide such as CRISPR-Cas9 can be lethal to the replicon targeted and can result in cell death rather than, for example, re- sensitisation to antibiotics, if an antibiotic resistance gene in the replicon is targeted for inactivation. Immediate cell death rather than such re-sensitisation to antibiotics for subsequent killing by antibiotic may increase selection pressure against delivery of the targeting constructs. Instead of DSBs, we may modify the Cas9 gene to allow the resealing of the targeted sequence after gene inactivation by introducing a deletion or inversion.

Tsai et al. (2014) have constructed a fusion between a catalytically inactive Cas9 (dCas9) protein to the wild-type nuclease domain of the restriction endonuclease Fok1 to increase the specificity of cleavage in eukaryotic cells. Proudfoot et al. (201 1 ) have similarly fused zinc- finger DNA recognition domains to the catalytic domains of recombinases to programme site- specific recombination at designated DNA sequences. We here add appropriate recombinase domains to dCas9 to catalyse a recombination reaction rather than a DSB at a CRISPR-mediated RNA-guided desired position on the target genes.

The Cas9 resolvase fusion, called Cas9R in Figure 18, is directed to the desired sites determined by the Cas9 domain :guide RNA spacer sequences; and the fused resolvase is positioned at the recombination site. To generate a deletion between two recombination sites: A and B, resolvases must dimerise at each recombination site. Thus two Cas9Rs need to be positioned in close proximity at each recombination site A1 A2 and B1 B2 as designated by the sequences encoded by the spacers S1 -S4. The correct orientations of A1 relative to A2 and B1 relative to B2 will need to be determined experimentally.

The orientation of the gRNA as shown Fig. 19 is already proved in the case of dCas9 fused Fok\ (Tsai el al., 2014) - i.e. PAM sequences of each protospacer sequence in the recombination sites are directly positioned at the outer boundaries of the full-length recombination site. Thus, we employ the same configuration of the annealed gRNA polarity.

Figures 19 and 20 show a schematic process as to how this Cas9R fusion resolvase resolves the synapse and parental replicon DNA is recircularised.

Example 7

The following experiments describe some proof-of-concept experiments performed to demonstrate that the CRISPR-Cas9 system can be used in a single construct to inactivate a large number of different beta-lactamase genes that may be found amongst microbial pathogens as well as amongst the non-pathogenic members of the microbiome.

In these exemplifications, plasmids are constructed that carry the CRISPR-Cas9 system plus derivatives carrying spacer sequences, flanked by direct repeats, targeted against up to eight of the following beta-lactamase families of resistance genes: SHV, CTX-M, TEM, KPC, VIM, IMP, NDM and OXA. For each of these eight families of beta-lactamase genes, a single spacer is designed that will target a number of gene members of that family. These are: SHV-a = 1 , 1 a, 2, 2a, 5, 5a, 1 1 , 12, 14, 18, 20, 21 , 22, 23, 26, 27, 28, 31 , 32, 33, 38, 43, 44, 48, 52, 55, 56, 60, 61 , 62, 71 , 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 85, 89, 92, 98, 99, 100, 101 , 103, 106, 107, 108, 109, 1 10, 1 1 1 , 121 , 136, 134, 137, 140, 143, 144, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 168, 172, 173, 178, 179; CTXM-b= 1 , 3, 10, 12, 15, 19, 22, 32, 52, 54, 59, 60, 62, 68, 71 , 80, 81 , 99, 141 , 147; TEM-c = 1 , 1 B, 3, 139, 162, 183, 192, 197, 198, 209; KPC-d = 1 , 2, 3, 4, 6, 7, 8, 1 1 , 12, 14, 15, 16, 17 ; VIM-e = 1 , 2, 4, 19, 26, 27, 33, 34; IMP-f = 4, 8, 32, 38; and NDM-g = 1 , 9, 10.

Figure 21 shows the eight spacer sequences that were designed to target the eight beta- lactamase families of resistance genes: SHV, CTX-M, TEM, KPC, VIM, IMP, NDM and OXA-48. The primer sequences used in the construction of the plasmids are listed in a table in Figure 22.

In one exemplification, a plasmid derivative of pNB100 (Figure 12), pNB104A (Figure 23), a generally applicable DNA cassette, is described in the Examples that carries the CRISPR- Cas9 system plus derivatives carrying spacer sequences, flanked by direct repeats, targeted against four beta-lactamase families of antibiotic resistance genes in bacteria and are expressed off one promotor: NDM, IMP, VIM and KPC.

In another exemplification, a plasmid derivative of pNB100, pNB104B (Figure 24), a generally applicable DNA cassette, is described in the Examples that carries the CRISPR- Cas9 system plus derivatives carrying spacer sequences, flanked by direct repeats, targeted against four beta-lactamase families of antibiotic resistance genes in bacteria and are expressed off one promotor: OXA-48, SHV, TEM and CTX-M.

In another exemplification, a plasmid derivative of pNB100, pNB108 (Figure 25), a generally applicable DNA cassette, is described in the Examples that carries the CRISPR- Cas9 system plus derivatives carrying spacer sequences, flanked by direct repeats, targeted against eight beta-lactamase families of antibiotic resistance genes in bacteria and are expressed off one promotor: SHV, CTX-M, TEM, KPC, VIM, IMP, NDM and OXA-48.

In another exemplification, a plasmid derivative of pNB100 is constructed where spacer sequences can be expressed from a choice of two different promotors. This plasmid, pNB200 (Figure 26), carries a different unique restriction site downstream of each promotor to clone any desired spacer sequences flanked by their own direct repeats between two direct repeats in the CRISPR locus. Derivatives of pNB200 are described that carry the CRISPR-Cas9 system plus derivatives carrying spacer sequences, flanked by direct repeats, targeted against eight beta-lactamase families of resistance genes: SHV, CTX-M, TEM, KPC, VIM, IMP, NDM and OXA-48. In pNB202 (Figure 27), the second promotor is used to express spacers flanked by direct repeats to target the four beta-lactamase families of resistance genes: OXA-48, SHV, TEM and CTX-M. And in pNB203 (Figure 28), which is derived from pNB202, both promotors are used: the first to express spacers flanked by direct repeats to target four beta-lactamase families of resistance genes: NDM, IMP, VIM and KPC and the second promotor to express spacers flanked by direct repeats to target four beta-lactamase families of resistance genes: OXA-48, SHV, TEM and CTX-M.

Construction of pNB104A

The tetramer spacer concatemer a+b+c+d shown in Figure 29A was digested with Bsa\, whose restriction site is located in A1 and A2, and ligated to Bsa\ spacer cloning sites on pNB100. The structure of the single promoter and spacer region (6221 -7001 ) on pNB104A is shown in Figure 23. The four-space concatemer contains spacer sequences targeting NDM, IMP, VIM and KPC from the proximal to the distal end of the single promoter.

Construction of pNB104B

The tetramer spacer sequence concatemer e+f+g+h shown in Figure 29A was digested with Sap\, whose restriction site is located in B1 and B2, and ligated to Sap\ spacer cloning sites on pNB200. While screening pNB202 screening this construct was found and confirmed by sequencing. A deletion event between the direct repeats adjacent to leader sequences had occurred and restored the direct repeat sequence to give pNB104B. The single promoter regions (6221 -6987) on pNB104B is shown in Figure 24. The concatenated spacers (targeted against OXA-48, SHV, TEM and CTX-M) are located under the single promoter.

Construction of pNB108

The concatenated spacer array sequences A and B were amplified from the subcloned vector pCR Blunt M-TOPO_SpacerA and pCR Blunt M-TOPO_SpacerB with the primer set NB026 and NB029, NB030 and NB033, respectively. At the 3' end of amplicon of spacer A and 5' end of amplicon of spacer B are 20 bases of overlapped sequence from KPC spacer sequence. These two amplicons were gel purified and used for PCR-based pairwise cycle extension reaction in the absence of the primer. The extended material was re-amplified with primer set NB037 (5'-GGGCTGGCAAGCCACGTTTGGTG-3'; SEQ ID NO. 150) and NB038 (5'-CCGGGAGCTGCATGTGTCAGAGG-3' ; SEQ ID NO. 151 ) to generate the full 8 spacer array concatemer. This eight-spacer concatemer was cloned into pCR Blunt II- TOPO vector and sequence was confirmed.

Bsa\ digestion of this pCR Blunt ll-TOPO subclone removes the full 8 spacer array concatemer as a subclone from the pCR Blunt ll-TOPO vector, which contains 5' protruding four base compatible bases on both ends for the sites created on pNB100 by Bsa\ digestion. Then pNB100 was digested with Bsa\ followed by agarose gel purification. The eight-spacer concatemer cassette, released from the pCR Blunt ll-TOPO was ligated into pNB100 by T4 DNA ligase and transformed to DH5a competent cells (purchased from New England Biolabs). The transformants were selected on chloramphenicol LB plates and were screened by PCR with the reverse primer NB021 : 5'- GGTGACTGATGGCCACGT (SEQ ID NO: 149) and a forward primer NB020: 5'- CCAACTACCTCCCCTTGCTTAAC (SEQ ID NO: 148), which anneal at 6368-6385 region and 7203-7225 region, respectively on the recombinant plasmid to generate 858 bp PCR amplicon. PCR-positive clones were sequenced to confirm the eight-spacer concatemer sequence and this recombinant clone is designated as pNB108 and used to demonstrate CRISPR/Cas9-mediated inactivation of targeted beta lactamase genes following DNA delivery to bacterial strains carrying such genes. A plasmid map of pNB108 is shown in Figure 25.

Construction of pNB200

The plasmid pNB200 contains two identical promotors for gRNA expression and carries a different unique restriction site Bsa\ and Sap\ downstream of each promotor to clone any desired spacer sequences flanked by their own direct repeats between two direct repeats in the CRISPR locus. The desired structure of the CRISPR array locus in pNB200 consists two sets of cassettes harbouring promotor-leader-direct repeat-spacer cloning region-direct repeat-tail tandemly. The forward primer NB018 anneals at the 5' end of the leader sequence to the promotor in pNB100 and introduces the first spacer cloning region, second direct repeat and tail sequence. The reverse primer NB019 anneals at the 3' end of leader sequence in pNB100 and introduces a third direct repeat, second spacer cloning region. This amplicon is cloned between Bsal sites on pNB100 to give pNB200. The amplified sequence (SEQ ID NO: 146) with primer NB018 and NB019 from pNB100 as template

5 'CCAAAACtgagacctGCTGCGGACGTCCAAAGGTCTCGTTTTAGAGCTATGCTGTTTTG AATGGTCCCAAAACTTG CCGATGATAACTTGAGAAAGAGGGTTAATACCAGCAGTCGGATACCTTCCTATTCTTTCT GTTAAAGCGTTTTCATGT TATAATAGGCAAATTTTAGATGAAGATTATTTCTTAATAACTAAAAATATGGTATAATAC TCTTAATAAATGCAGTAA TACAGGGGCTTTTCAAGACTGAAGTCTAGCTGAGACAAATAGTGCGATTACGAAATTTTT TAGACAAAAATAG TCTAC GAGGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAACtGAAGAGCGTCTCGGACgcaL qcGCTCT TCGTTTTAGA . The forward primer NB018 and the reverse primer NB019 are underlined and the initial annealing sites, for the first PCR cycle, are indicated by bold letters and italicised bold letters, respectively. This amplicon was digested with Bbv (5'-GCAGCN8/N12) to create four bases compatible protruding 5' ends to Bsa\ digested pNB100. Bbv\ recognition sites are depicted by lower bold cases.

The PCR conditions to generate the dual promotor-leader cassette were:

Cycle conditions

72 °C 30 sec

Final Extension 72 °C 10 min

The sequence of the modified CRISPR array in pNB200 (SEQ ID NO: 147) The total number of nucleotides of pNB200 is 9919 bp. The sequence in the modified region is shown below, which replaces the small Bsa\ fragment in pNB100. The first and the second promotor sequences are underlined. Leader sequences are in bold case. Direct repeats are italicised and the spacer cloning region between direct repeats are indicated by italicised and underlined.

The first spacer array cloning region contains two inverted Bsa\ sites indicated by bold italicised underlined letters 5'-GAGACC-3' and 5'-GGTCTC-3' for creating 5' four bases protruding spacer cloning sites 5'-GTTT-3' and 5'-TTTT-3' on the vector, respectively and one unique Aati\ (5'-GACGTC-3') site also indicated by bold italicised underlined lettes to reduce self-ligation in the event of incomplete Bsa\ digestion.

The second spacer cloning region contains two inverted Sap\ sites indicated by bold italicised underlined letters 5'-GAAGAGC-3' and 5'-GCTCTTC-3' for creating 5' three bases protruding spacer cloning sites 5'-GTT-3' and 5'-TTT-3' on the vector, respectively.

GGTGACTGATGGCCACGTGAACTATATGATTTTCCGCAGTATATTTTAGATGAAGAT TATTTCTTAATAACTAAAAAT ATGGTATAATACTCTTAATAAATGCAGTAATACAGGGGCTTTTCAAGACTGAAGTCTAGC TGftGftCAAATAGTGCGftT TACGAAATTTTTTAGACAAAAATAGTCTACGAGG TTTTAGAGCTATGCTATTTTGAATGGTCCCAAAACtGAGACCtG CTGCGGACGTCCAAAGGTCTCGTTTTAGAGCTATGCTGTTTTGAATGGTCCCAAAACttg ccgatgataacttgagaa agagggttaataccagcagtcggataccttcctattctttctgttaaagcgttttcatgt tataataggcaaATTTTA GATGAAGATTATTTCTTAATAACTAAAAATATGGTATAATACTCTTAATAAATGCAGTAA TACAGGGGCTTTTCAAGA CTGAAGTCTAGCTGAGACAAATAGTGCGATTACGAAATTTTTTAGACAAAAATAGTCTAC GAGGrrrrAGAGCrATGC TGTTTTGAATGGTCCCAAAACtGAAGAGCGTCTCGGACGCAGCGCTCTTCGTTTTAGAGC TATGCTGTTTTGAATGGT CCCAAAACaacattgccgatgataacttgagaaagagggttaataccagcagtcggatac cttcctattctttctgtt aaagcgttttcatgttataataggcaaaagaagagtagtgtg

A plasmid map of pNB200 is shown in Figure 26. The desired spacer array sequences can be cloned in the clockwise direction between two Bsa\ sites and two Sap\ sites

independently, for each promotor respectively. Construction of pNB202

The plasmid pNB201 contains the dual promotors of pNB200 from which it is derived but a spacer array sequence targeted to OXA-48, SHV, TEM and CTX-M beta lactamase gene families are expressed from the second promotor.

To construct pNB202, pNB200 was digested with Bsa\ followed by agarose gel purification. The concatenated spacer array sequence B was generated by PCR-mediated pair-wise oligo nucleotide concatenation. This four-spacer concatemer array B was cloned to the pCR Blunt ll-TOPO vector, purchased from Life Technologies, and the sequence was confirmed. Sap\ cuts out spacer array sequence B as a subclone from the TOPO vector and yields 5' protruding three base compatible bases on both ends for the sites created on pNB200 by Sap\ digestion. This four-spacer concatemer B cassette was ligated to pNB200 by T4 DNA ligase and transformed to DH5a competent cells (purchased from New England Biolabs). The transformants were selected on chloramphenicol LB plate and were screened by PCR with the reverse primer NB021 : 5'- GGTGACTGATGGCCACGT (SEQ ID NO: 149) and a forward primer NB020: 5'- CCAACTACCTCCCCTTGCTTAAC (SEQ ID NO: 148), which anneal at 6368-6385 region and 7307-7329 region, respectively on the recombinant plasmid to generate 962 bp PCR amplicon. PCR positive clones were sequenced to confirm the four-spacer concatemer B sequence and this recombinant clone is designated as pNB202 and used to demonstrate CRISPR/Cas9-mediated inactivation of targeted beta lactamase genes following DNA delivery to bacterial strains carrying such genes. A plasmid map of pNB202 is shown in Figure 27.

Construction of pNB203

The plasmid pNB203 contains the dual promotors of pNB200 but each promotor expresses four spacers. The first promotor expresses spacer sequence targeted to NDM, IMP, VIM and KPC, the second promotor expresses OXA, SHV, TEM and CTX-M beta lactamase genes. The plasmid pNB202 was digested with Bsa\ followed by agarose gel purification. The concatenated spacer array sequence A was cut out from pCR Blunt ll-TOPO vector harbouring spacer concatemer A with Bsa\. Bsa\ cuts out spacer region, which contains 5' protruding four base compatible bases on both ends for the sites created on pNB202 by Bsa\ digestion. This four-spacer concatemer A cassette was ligated to pNB202 by T4 DNA ligase and transformed to DH5a competent cells (purchased from New England Biolabs). The transformants were selected on chloramphenicol LB plate and were screened by PCR with the reverse primer NB021 : 5'- GGTGACTGATGGCCACGT and a forward primer NB020: 5'- CCAACTACCTCCCCTTGCTTAAC , which anneal at 6368-6385 region and 7479-7501 region, respectively on the recombinant plasmid to generate 1 134 bp PCR amplicon. PCR positive clones were sequenced to confirm the four-spacer concatemer A and B sequence and this recombinant clone is designated as pNB203 and used to demonstrate CRISPR/Cas9-mediated inactivation of targeted beta lactamase genes following DNA delivery to bacterial strains carrying such genes. A plasmid map of pNB203 is shown in Figure 28.

Schematic representation of the structure of concatenated spacer arrays

Spacer sequences were determined to maximise the coverage of the target beta-lactamase gene family. Each unit oligo contains the direct repeat flanking the appropriate spacer sequence at each end. Concatenation reactions are performed between pairwise oligos, i.e. the nearest neighbour unit oligos are concatenated first to generate two unit length oligo, then two unit length oligos are concatenated to generate four unit length of oligo etc.

The schematic structure of tetramer and octamer spacer structures are shown in Figure 29. S: spacer, R: direct repeat, A and B contain Bsa\ site to create ligation compatible sites for cloning into pNB100 and downstream of the first promotor of pNB200. C and D contain a Sap\ site to create ligation a compatible site for cloning downstream of the second promotor of pNB200. In this example, we employed spacer sequence S1 targeting NDM, S2 targeting IMP, S3 targeting VIM, S4 targeting KPC, S5 targeting OXA, S6 targeting SHV, S7 targeting TEM and S8 targeting CTX-M.

Spacer concatenation reaction

Each oligo has overlapped sequence in the 3' and 5' end to anneal to the nearest neighbour oligo except the first and the last oligo. The first and the last oligo have the overlapping sequence to the second and the penultimate oligo in the 5' end only. In order to concatenate four spacers, four oligos are synthesised. In other words, oligo No.1 consists spacer 1 and 2 in the 5' and 3' ends. Oligo No.2 contains reverse complement of spacer 2 and 3 in the 3' and 5' ends. Oligo No.3 contains spacer 3 and 4 in the 5' and 3' ends. Oligo No.4 contains reverse complement of spacer 4 in the 3' end. Thus the oligo No.2 can link oligo No.1 and oligo No.3, oligo 4 anneals to 3' end of oligo No.3. Oligo No.1 and oligo No. 2, oligo No. 3 and oligo No. 4 are concatenated in a separate tube using the following PCR reaction conditions. Component A1 Α2 Β1 Β2 χ 4

Nuclease-Free water 33.5 μΙ_ 33.5 μΙ_ 33.5 μΙ_ 33.5 μΙ_ 134

5X Q5 Reaction Buffer 10 μΙ_ 10 μΙ_ 10 μΙ_ 10 μΙ_ 40

10 mM dNTPs 1 μί. 1 μΐ 1 μΐ 1 μΐ 4

Q5 High-Fidelity DNA Polymerase 0.5 μΙ_ 0.5 μΙ_ 0.5 μΙ_ 0.5 μΐ 2

10 μΜ Forward Primer NB026 2.5 μΙ_

10 μΜ Reverse Primer NB027 2.5 μΙ_

10 μΜ Forward Primer NB028 2.5 μΙ_

10 μΜ Reverse Primer NB034 2.5 μΙ_

10 μΜ Forward Primer NB035 2.5 μΙ_

10 μΜ Reverse Primer NB031 2.5 μΙ_

10 μΜ Forward Primer NB032 2.5 μΙ_

10 μΜ Reverse Primer NB036 2.5 μί

Cycle conditions

In this example, NB026 and NB027, NB028 and NB034, NB035 and NB031 , NB032 and NB036 are concatenated. Each concatenated product A1 , A2, B1 and B2 was gel purified and set up the second concatenation reaction using the purified A1 and A2, B1 and B2 dimer product in the following PCR condition.

Cycle conditions

These extention products were amplified by NB037 and NB038 with Q5 DNA polym The final amplicons were cloned to pCR Blunt II TOPO vector and the concatemer sequences were confirmed. In case of eight spacer concatenation, spacer concatemer A and spacer concatemer B on pCR Blunt II TOPO vector were amplified with primer pairs NB026 and NB029, NB030 and NB033, respectively and amplicons were gel purified. Purified spacer A and B were utilised as a long primer in the following cycle extension reaction.

Cycle conditions

These extention products were amplified by NB037 and NB038 with Q5 DNA polymerase. The final amplicons were cloned into pCR Blunt II TOPO vector and the concatemer sequences were confirmed. Delivery of CRISPR-Cas9 constructs to bacteria

Constructs that are able to inactivate target genes, including antibiotic resistant genes, via the CRISPR-Cas system, and which are an aspect of the present invention are also referred to herein as "Nemesis symbiotics". The CRISPR-Cas9 plasmid derivatives, pNB104A, pNB104B, pNB202 and pNB203 all carry spacer insertions targeted against the selected families of beta-lactamase (bla) genes described, and so provide exemplars to demonstrate that a single plasmid construct possesses Nemesis symbiotic activity (NSA) and is therefore able to inactivate representative genes from all 8 different families of beta- lactam antibiotics. The plasmid pNB104A (see Fig. 23) is tested for its ability to inactivate representative genes members of the NDM, IMP, VIM and KPC families; pNB104B (see Fig. 24) and pNB202 (ss Fig. 27) are tested for their ability to inactivate representative genes members of the OXA, SHV, TEM and CTX-M families; and pNB108 (see Fig. 25) and pNB203 (see Fig. 28) are tested for their ability to inactivate representative genes members of the SHV, CTX-M, TEM, KPC, VIM, IMP, NDM and OXA families.

Nemesis symbiotic activity (NSA) assay by plasmid transformation

The NSA assay described in Example 2 showed that DNA transformation of an E. coli strain, DH5a, also carrying the TEM-3 beta lactamase gene on the plasmid pBR322, with plasmid pNB102 converts the transformant to ampiciilin sensitivity (ApS). Here, the plasmid pNB102 encodes resistance to chloramphenicol and the DH5a (pBR322) transformants now carrying pNB102 were selected on LB Cm plates and then screened for ApS (see Fig. 14). Here, the plasmid pNB102, in expressing the CRISPR/Cas9 system with the spacer sequence encoding the gRNA targeting the TEM-3 gene, inactivated the TEM-3 gene. In contrast in a negative control experiment, when DH5a (pBR322) was transformed with the parental plasmid pNB100 carrying the expressing the CRISPR/Cas9 system but lacking the gRNA targeting the TEM-3 gene, no conversion to ApS occurred.

For exemplification purposes an equivalent experiment is described where plasmid derivatives of pBR322 are constructed where the TEM-3 is replaced by representative genes from the other 7 different families of beta-lactam antibiotics: SHV, CTX-M, KPC, VIM, IMP, NDM and OXA. Such genes are obtained from suitable bacterial strains carrying such genes. This allows a direct comparison to the proof of concept experiments described in Example 2 in isogenic genetic backgrounds. A set of E. coli and K. pneumoniae strains carrying representative genes from these seven different families of beta-lactam antibiotics were purchased from Culture Collections, Public Health England, Porton Down, Salisbury, SP4 OJG, UK. These are: NCTC13368, a K. pneumoniae strain carrying the SHV-18 gene; NCTC13353 an E. coli strain carrying the CTX-M-15 gene; NCTC13438 a K. pneumoniae strain carrying the KPC-3 gene;

NCTC13440 a K. pneumoniae strain carrying the VIM-1 gene; NCTC13476 an E. coli strain carrying an uncharacterised IMP gene; NCTC13443 a K. pneumoniae strain carrying the NDM-1 gene and NCTC13442 a K. pneumoniae strain carrying the OXA-48 gene. All seven genes encode beta lactamases that are also able to degrade and inactivate the penam class of antibiotics (see Fig. 16, 17). All strains were tested and, as expected, found to be resistant to the penam class antibiotic, ampicillin.

Beta lactamase coding sequences are amplified from the cell with appropriate forward and reverse primer set shown below:

Resist:ance

Strain HCTC No. Forward primer 5' to 3 ' Reverse primer 5' to 3' gene

NBKpDOl 13443 NDM-1 attgaaaaaggaagagtATGGAATTGCCC agtcccgctaGGTCTCaACCGTCAGCGCA

AATATTATGCACCC (SEQ ID NO: GCTTGTCGG (SEQ ID NO : 153) 152)

NBKp002 13442 OXA-48 attgaaaaaggaagagtATGCGTGTATTA agtcccgctaGGTCTCaACCGCTAGGGAA

GCCTTATCGGCTG {SEQ ID NO: TAATTTTTTCCTGTTTGAGCACTTCT

154) (SEQ ID NO: 155)

NB p003 13368 SHV-18 attgaaaaaggaagagtATGCGTTATTTT agtcccgctaGGTCTCaACCGTTAGCGTT

CGCCTGTGTATTATCTCC (SEQ ID GCCAGTGCTCGA {SEQ ID NO: 157) NO: 156)

NBKpOQ4 13440 VIM-1 attgaaaaaggaagagtATGTTAAAAGTT agtcccgctaGGATGacctggctgACCGC

ATTAGTAGTTTATTGGTCTACATGACCG TACTCGGCGACTGAGCGAT {SEQ ID

{SEQ ID NO: 158) NO: 159)

BKpOOS 13438 KPC-3 attgaaaaaggaagagtATGTCACTGTAT agtcccgctaGGTCTCaACCGTTACTGCC

CGCCG CTAGTTCT (SEQ ID NO: CGTTGACGCC {SEQ ID NO: 161)

ISO)

NBEcOlS 13476 IMP-4 attgaaaaaggaagagtATGAGCAAGTTA agtcccgctaGGTCTCaACCGTTAGTTGC

TCTGTATTCTTTATATTTTTGTTTTGTAG TTAGTTTTGATGGTTTTTTACTTTCGTTT

CA (SEQ ID NO: 162) ARC {SEQ ID NO: 163)

NBEc019 13353 CTX-M-15 attgaaaaaggaagagtATGGTTAAAAAA agtcccgctaGGTCTCaACCGTTACAAAC

TCACTGCGCCAGTTC (SEQ ID NO: CGTCGGTGACGATTTTAG (SEQ ID NO:

164) 165)

Each forward primer contains a 17 base sequence to restore the beta-lactamase promoter on pBR322, and each reverse primer contains Bsa\ site (for NDM-1 , OXA-48, SHV-18, KPC-3, IMP-4 and CTX-M15) or Fok\ site (for VIM-1 ) to create 5'-ACCG four base protruding 5' end. After amplifying each beta lactamase gene with high fidelity DNA polymerase such as Q5 DNA polymerase, the amplicon is digested with the appropriate restriction enzyme located in the reverse primer, described above. The digested amplicons are ready to ligate using T4 ligase between the Ssp\ and Bsa\ sites on the plasmid pBR322 (purchased from New England Biolabs), after removal of the TEM-3 fragment. Ssp\ creates a blunt end and Bsa\ creates a 5'-CGGT protruding end. The reverse complement of the coding sequences of the each amplicons after restriction digestion are shown below. The 5' protruding end is underlined and 3' end of the promotor sequence is in bold small letters. The reverse complement CAT of the methionine initiating codons ATG of these seven genes, also shown in bold, yields a precise fusion of the coding region of the seven other beta-lactamases to the translational signal sequences of the TEM-3 beta-lactamase of pBR322.

NDM-1 (SEQ ID NO: 166)

ACCGTCAGCGCAGCTTGTCGGCCATGCGGGCCGTATGAGTGATTGCGGCGCGGCTATCGG GGGCGGA ATGGCTCATCACGATCATGCTGGCCTTGGGGAACGCCGCACCAAACGCGCGCGCTGACGC GGCGTAG TGCTCAGTGTCGGCATCACCGAGATTGCCGAGCGACTTGGCCTTGCTGTCCTTGATCAGG CAGCCAC CAAAAGCGATGTCGGTGCCGTCGATCCCAACGGTGATATTGTCACTGGTGTGGCCGGGGC CGGGGTA AAATACCTTGAGCGGGCCAAAGTTGGGCGCGGTTGCTGGTTCGACCCAGCCATTGGCGGC GAAAGTC AGGCTGTGTTGCGCCGCAACCATCCCCTCTTGCGGGGCAAGCTGGTTCGACAACGCATTG GCATAAG TCGCAATCCCCGCCGCATGCAGCGCGTCCATACCGCCCATCTTGTCCTGATGCGCGTGAG TCACCAC CGCCAGCGCGACCGGCAGGTTGATCTCCTGCTTGATCCAGTTGAGGATCTGGGCGGTCTG GTCATCG GTCCAGGCGGTATCGACCACCAGCACGCGGCCGCCATCCCTGACGATCAAACCGTTGGAA GCGACTG CCCCGAAACCCGGCATGTCGAGATAGGAAGTGTGCTGCCAGACATTCGGTGCGAGCTGGC GGAAAAC CAGATCGCCAAACCGTTGGTCGCCAGTTTCCATTTGCTGGCCAATCGTCGGGCGGATTTC ACCGGGC ATGCACCCGCTCAGCATCAATGCAGCGGCTAATGCGGTGCTCAGCTTCGCGACCGGGTGC ATAATAT TGGGCAATTCCATactcttcctttttcaat

OXA-48 (SEQ ID NO: 167)

ACCGCTAGGGAATAATTTTTTCCTGTTTGAGCACTTCTTTTGTGATGGCTTGGCGCAGCC CTAAACC ATCCGATGTGGGCATATCCATATTCATCGCAAAAAACCACACATTATCATCAAGTTCAAC CCAACCG ACCCACCAGCCAATCTTAGGTTCGATTCTAGTCGAGTATCCAGTTTTAGCCCGAATAATA TAGTCAC CATTGGCTTCGGTCAGCATGGCTTGTTTGACAATACGCTGGCTGCGCTCCGATACGTGTA ACTTATT GTGATACAGCTTTCTTAAAAAGCTGATTTGCTCCGTGGCCGAAATTCGAATACCACCGTC GAGCCAG AAACTGTCTACATTGCCCGAAATGTCCTCATTACCATAATCGAAAGCATGTAGCATCTTG CTCATAC GTGCCTCGCCAATTTGGCGGGCAAATTCTTGATAAACAGGCACAACTGAATATTTCATCG CGGTGAT TAGATTATGATCGCGATTCCAAGTGGCGATATCGCGCGTCTGTCCATCCCACTTAAAGAC TTGGTGT TCATCCTTAACCACGCCCAAATCGAGGGCGATCAAGCTATTGGGAATTTTAAAGGTAGAT GCGGGTA AAAATGCTTGGTTCGCCCGTTTAAGATTATTGGTAAATCCTTGCTGCTTATTCTCATTCC AGAGCAC AACTACGCCCTGTGATTTATGTTCAGTAAAGTGAGCATTCCAACTTTTGTTTTCTTGCCA TTCCTTT GCTACCGCAGGCATTCCGATAATCGATGCCACCAAAAACACAGCCGATAAGGCTAATACA CGCATac tcttcctttttcaat

SHV-18 (SEQ ID NO: 168)

ACCGTTAGCGTTGCCAGTGCTCGATCAGCGCCGCGCCGATCCCGGCGATTTGCTGATTTC GCTCGGC CATGCTCGCCGGCGTATCCCGCAGATAAATCACCACAATCCGCTCTGCTTTGTTATTCGG GCCAAGC AGGGCGACAATCCCGCGCGCACCCCGTTTGGCAGCTCCGGTCTTATCGGCGATAAACCAG CCCGCCG GCAGCACGGAGCGGATCAACGGTCCGGCGACCCGATCGTCCACCATCCACTGCAGCAGCT GCCGTTG CGAACGGGCGCTCAGACGCTGGCTGGTCAGCAGCTTGCGCAGGGTCGCGGCCATGCTGGC CGGGGTA GTGGTGTCGCGGGCGTCGCCGGGAAGCGCCTCATTCAGTTCCGTTTCCCAGCGGTCAAGG CGGGTGA CGTTGTCGCCGATCTGGCGCAAAAAGGCAGTCAATCCTGCGGGGCCGCCGACGGTGGCCA GCAGCAG ATTGGCGGCGCTGTTATCGCTCATGGTAATGGCGGCGGCACAGAGTTCGCCGACCGTCAT GCCGTCG GCAAGGTGTTTTTCGCTGACCGGCGAGTAGTCCACCAGATCCTGCTGGCGATAGTGGATC TTTCGCT CCAGCTGTTCGTCACCGGCATCCACCCGCGCCAGCACTGCGCCGCAGAGCACTACTTTAA AGGTGCT CATCATGGGAAAGCGTTCATCGGCGCGCCAGGCGGTCAGCGTGCGGCCGCTGGCCAGATC CATTTCT ATCATGCCTACGCTGCCCGACAGCTGGCTTTCGCTTAGTTTAATTTGCTCAAGCGGCTGC GGGCTGG CGTGTACCGCCAGCGGCAGGGTGGCTAACAGGGAGATAATACACAGGCGAAAATAACGCA Tactctt cctttttcaat

VIM-1 (SEQ ID NO: 169) ACCGCTACTCGGCGACTGAGCGATTTTTGTGTGCTTTGACAACGTTCGCTGTGTGCTGGA GCAAGTC TAGACCGCCCGGTAGACCGTGCCCGGGAATGACGACCTCTGCTTCCGGGTAGTGTTTTTG AATCCGC TCAACGGAGGTGGGCCATTCAGCCAGATCGGCATCGGCCACGTTCCCCGCAGACGTGCTT GACAACT CATGAACGGCACAACCACCGTATAGCACGTTCGCTGACGGGACGTATACAACCAGATTGT CGGTCGA ATGCGCAGCACCAGGATAGAAGAGCTCTACTGGACCGAAGCGCACTGCGTCCCCGCTCGA TGAGAGT CCTTCTAGAGAATGCGTGGGAATCTCGTTCCCCTCTGCCTCGGCTAGCCGGCGTGTCGAC GGTGATG CGTACGTTGCCACCCCAGCCGCCCGAAGGACATCAACGCCGCCGACGCGGTCGTCATGAA AGTGCGT GGAGACTGCACGCGTTACGGGAAGTCCAATTTGCTTTTCAATCTCCGCGAGAAGTGCCGC TGTGTTT TTCGCACCCCACGCTGTATCAATCAAAAGCAACTCATCACCATCACGGACAATGAGACCA TTGGACG GGTAGACCGCGCCATCAAACGACTGCGTTGCGATATGCGACCAAACACCATCGGCAATCT GGTAAAG TCGGACCTCTCCGACCGGAATTTCGTTGACTGTCGGATACTCACCACTCGGCTCCCCGGA ATGGGCT AACGGACTTGCGACAGCCATGACAGACGCGGTCATGTAGACCAATAAACTACTAATAACT TTTAACA

Tactcttcctttttcaat

KPC-3 (SEQ ID NO: 170)

ACCGTTACTGCCCGTTGACGCCCAATCCCTCGAGCGCGAGTCTAGCCGCAGCGGCGATGA CGGCCTC GCTGTACTTGTCATCCTTGTTAGGCGCCCGGGTGTAGACGGCCAACACAATAGGTGCGCG CCCAGTG GGCCAGACGACGGCATAGTCATTTGCCGTGCCATACACTCCGCAGGTTCCGGTTTTGTCT CCGACTG CCCAGTCTGCCGGCACCGCCGCGCGGATGCGGTGGTTGCCGGTCGTGTTTCCCTTTAGCC AATCAAC AAACTGCTGCCGCTGCGGCGCAGCCAGTGCAGAGCCCAGTGTCAGTTTTTGTAAGCTTTC CGTCACG GCGCGCGGCGATGAGGTATCGCGCGCATCGCCTGGGATGGCGGAGTTCAGCTCCAGCTCC CAGCGGT CCAGACGGAACGTGGTATCGCCGATAGAGCGCATGAAGGCCGTCAGCCCGGCCGGGCCGC CCAACTC CTTCAGCAACAAATTGGCGGCGGCGTTATCACTGTATTGCACGGCGGCCGCGGACAGCTC CGCCACC GTCATGCCTGTTGTCAGATATTTTTCCGAGATGGGTGACCACGGAACCAGCGCATTTTTG CCGTAAC GGATGGGTGTGTCCAGCAAGCCGGCCTGCTGCTGGCTGCGAGCCAGCACAGCGGCAGCAA GAAAGCC CTTGAATGAGCTGCACAGTGGGAAGCGCTCCTCAGCGCGGTAACTTACAGTTGCGCCTGA GCCGGTA TCCATCGCGTACACACCGATGGAGCCGCCAAAGTCCTGTTCGAGTTTAGCGAATGGTTCC GCGACGA GGTTGGTCAGCGCGGTGGCAGAAAAGCCAGCCAGCGGCCATGAGAGACAAGACAGCAGAA CTAGACG GCGATACAGTGACATactcttcctttttcaat

IMP-4 (SEQ ID NO: 171 )

ACCGTTAGTTGCTTAGTTTTGATGGTTTTTTACTTTCGTTTAACCCTTTAACCGCCTGCT CTAATGT AAGTTTCAAGAGTGATGCGTCTCCAGCTTCACTGTGACTTGGAACAACCAGTTTTGCCTT ACCATAT TTGGATATTAATAATTTAGCGGACTTTGGCCAAGCTTCTAAATTTGCGTCACCCAAATTA CCTAGAC CGTACGGTTTAATAAAACAACCACCGAATAATATTTTCCTTTCAGGCAGCCAAACTACTA GGTTATC TGGAGTGTGTCCTGGGCCTGGATAAAAAACTTCAATTTTATTTTTAACTAGCCAATAGTT AACCCCG CCAAATGAATTTTTAGCTTGAACCTTACCGTCTTTTTTAAGCAGCTCATTAGTTAATTCA GACGCAT ACGTGGGGATGGATTGAGAATTAAGCCACTCTATTCCGCCCGTGCTGTCACTATGAAAAT GAGAGGA AATACTGCCTTTTATTTTATAGCCACGTTCCACAAACCAAGTGACTAACTTTTCAGTATC TTTAGCC GTAAATGGAGTGTCAATTAGATAAGCTTCAGCATCTACAAGAACAACCAAACCATGTTTA GGAACAA CGCCCCACCCGTTAACTTCTTCAAACGAAGTATGAACATAAACGCCTTCATCAAGTTTTT CAATTTT TAAATCTGGCAAAGACTCTGCTGCGGTAGCAATGCTACAAAACAAAAATATAAAGAATAC AGATAAC TTGCTCATactcttcctttttcaat

CTX-M-15 (SEQ ID NO: 172)

ACCGTTACAAACCGTCGGTGACGATTTTAGCCGCCGACGCTAATACATCGCGACGGCTTT CTGCCTT AGGTTGAGGCTGGGTGAAGTAAGTGACCAGAATCAGCGGCGCACGATCTTTTGGCCAGAT CACCGCG ATATCGTTGGTGGTGCCATAGCCACCGCTGCCGGTTTTATCCCCCACAACCCAGGAAGCA GGCAGTC CAGCCTGAATGCTCGCTGCACCGGTGGTATTGCCTTTCATCCATGTCACCAGCTGCGCCC GTTGGCT GTCGCCCAATGCTTTACCCAGCGTCAGATTCCGCAGAGTTTGCGCCATTGCCCGAGGTGA AGTGGTA TCACGCGGATCGCCCGGAATGGCGGTGTTTAACGTCGGCTCGGTACGGTCGAGACGGAAC GTTTCGT CTCCCAGCTGTCGGGCGAACGCGGTGACGCTAGCCGGGCCGCCAACGTGAGCAATCAGCT TATTCAT CGCCACGTTATCGCTGTACTGTAGCGCGGCCGCGCTAAGCTCAGCCAGTGACATCGTCCC ATTGACG TGCTTTTCCGCAATCGGATTATAGTTAACAAGGTCAGATTTTTTGATCTCAACTCGCTGA TTTAACA GATTCGGTTCGCTTTCACTTTTCTTCAGCACCGCGGCCGCGGCCATCACTTTACTGGTGC TGCACAT CGCAAAGCGCTCATCAGCACGATAAAGTATTTGCGAATTATCTGCTGTGTTAATCAATGC CACACCC AGTCTGCCTCCCGACTGCCGCTCTAATTCGGCAAGTTTTTGCTGTACGTCCGCCGTTTGC GCATACA GCGGCACACTTCCTAACAACAGCGTGACGGTTGCCGTCGCCATCAGCGTGAACTGGCGCA GTGATTT TTTAACCATactcttcctttttcaat Then DH5a competent cells purchased from New England Biolabs are transformed with these ligations followed by selection for the desired recombinants on LB Ampicillin (100^g/ml_) plates.

Plasmid DNA samples are isolated from these transformants and submitted to DNA sequence analysis to confirm that the correct sequence for each of the seven different beta lactamases genes is present in each construct giving the plasmids:

These pBR322 derivative plasmids so derived are named:

i. pNB010 carrying the SHV-18 gene;

ii. pNB01 1 carrying the CTX-M-15 gene;

iii. pNB012 carrying the KPC-3 gene;

iv. pNB013 carrying the VIM-1 gene;

v. pNB014 carrying the IMP gene;

vi. pNB015 carrying the NDM-1 gene;

vii. pNB016 carrying the OXA-48 gene; in addition to, as described,

viii. pBR322 carrying the TEM-3 gene

Then 7 recipient E. coli strains, DH5a, each carrying one of these seven beta lactamase genes on the plasmids pNB010-016 are subsequently transformed with the plasmids pNB104A, pNB104B, pNB108 and pNB203 and selected for chloramphenicol resistance to select for acquisition of these Nemesis symbiotic plasmids, along with the negative control pNB100 as well as with transformation of DH5a (pBR322) by pNB102 as the positive control, and then tested for conversion to ampicillin sensitivity as described in Example 2 (see Fig 14.)

"Nemesis symbiotic activity" (NSA) assay by plasmid conjugation to test activity of constructs carrying multiple spacers targeting beta lactamase gene families

The plasmid conjugation assay described in Example 2 may also be used to test the Nemesis symbiotic activity of the new CRISPR/Cas9 plasmid constructs carrying spacer sequences targeting multiple families of beta lactamase genes. The assay involves mating a donor cell carrying the beta lactamase gene on a conjugative plasmid, and hence ampicillin resistant, with a recipient cell carrying the Nemesis symbiotic on a non- mobilisable plasmid encoding chloramphenicol resistance. Exconjugants are selected on LB plates containing both 100^g/ml_ ampicillin and 35μg/mL chloramphenicol. Successful Nemesis symbiotic activity is seen in a reduction in the efficiency of transfer of the ampicillin resistance gene.

As donors, the same strain used in Example 2 was used. This strain, JA200 (F+ thr-1 , leu- 6, DE(trpE)5, recA, lacY, thi, gal, xyl, ara, mtl), also carries plasmid pNT3 as described by Saka et al. (DNA Research 12, 63-68, 2005). The plasmid pNT3 is a mobilisable plasmid carrying the TEM-1 beta lactamase gene of Tn1 . Conjugation of pNT3 and hence transfer of ampicillin resistance is effected by the transfer functions of the co-resident F+ plasmid.

The other potential donors are the set of E. coli and K. pneumoniae strains carrying representative genes from these seven different families of beta-lactam antibiotics that were purchased from Culture Collections, Public Health England, as described above. These strains need to be chloramphenicol sensitive in order to allow selection for exconjugants and were tested for growth on LB chloramphenicol 35μg/mL plates.

NCTC13368, a K. pneumoniae strain carrying the SHV-18 gene; NCTC13438 a K.

pneumoniae strain carrying the KPC-3 gene; NCTC13476 an E. coli strain carrying an uncharacterised IMP gene; NCTC13443 a K. pneumoniae strain carrying the NDM-1 gene and NCTC13442 a K. pneumoniae strain carrying the OXA-48 gene were all found to be resistant to chloramphenicol, but NCTC13353, an E. coli strain carrying the CTX-M-15 gene, and NCTC13440, a K. pneumoniae strain carrying the VIM-1 gene, were found to be chloramphenicol sensitive and were taken forward to be tested in matings with the recipients.

As recipients, the strains used in Example 2 serve as controls for testing the new plasmid constructs. Thus the negative control is DH5a (F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG 08OdlacZAM15 A(lacZYA-argF)U169, hsdR17(rK- mK+), λ with the plasmid pNB100 encoding the CRISPR/Cas9 cassette but no spacer sequence targeting any antibiotic resistance gene; and the positive control is DH5a with the plasmid pNB102 encoding the CRISPR/Cas9 cassette as well as the spacer sequence targeting the TEM beta-lactamase family. To be tested are DH5a strains with the plasmids pNB104A, pNB104B and pNB108. These plasmids also encode the CRISPR/Cas9 cassette in addition to the spacer sequences, all driven off one promotor in the following order, proximal to distal from the promotor (pNB104A): NDM-IMP-VIM-KPC; (pNB104B): OXA- 48-SHV-TEM-CTX-M (pNB108): NDM-IMP-VIM-KPC-OXA-48-SHV-TEM-CTX-M. To set up the matings, colonies of donors were picked from LB Ap100 (ampicillin

100μg/mL) and recipients from LB Cm35 (chloramphenicol 35μg/mL) plates into 200μί of LB and then mixed 10Ομί of recipients with 35μί of donors, then 5μί of the mixture were spotted onto LB plates and incubated at 37 ² C for 5 hours to allow mating.

Figure 30A shows the results from mating the donor carrying the TEM-1 beta lactamase (JA200 (F+ thr-1 , leu-6, DE(trpE)5, recA, lacY, thi, gal, xyl, ara, mtl)), and designated (5) in the figure, with the recipients DH5a pNB100, designated (-), DH5a pNB102, designated (+). Here a loopful of each mating mixture was resuspended in 220μί of LB and 200μί of the cells were plated on LB Ap100Cm35 plates to select for exconjugants. As expected from Example 2, the cross between the donor of TEM-1 and the recipient with pNB100 gives efficient mating and a lawn of cells (Fig. 30A, top left, 5-), and mating the donor of TEM-1 with the recipient with pNB102 encoding the single TEM spacer shows strong inhibition of transfer of the TEM-1 gene (see Fig. 30A, 5+, top right, where only a few isolated colonies are seen). The mating of the donor carrying the TEM-1 gene with the recipient carrying pNB104 (see bottom plate, 5B, of the figure) also shows strong inhibition of transfer of the TEM-1 gene and demonstrates that in the pNB104B construct, carrying the four spacers OXA-48-SHV-TEM-CTX-M 4, the TEM spacer is active. Figure 30B shows the results from mating the strain NCTC13440, a K. pneumoniae strain carrying the VIM-1 , designated (2), and the strain NCTC13353, an E. coli strain carrying the CTX-M-15 gene, designated (4). Again a loopful of each such mating mixture was resuspended in 220μί of LB and 200μί of the cells were plated on LB Ap100Cm35 plates to select for exconjugants. The results show that these strains are able to act as donors and transfer the VIM-1 and the CTX-M-15 genes respectively to the DH5a pNB100 negative control recipient lacking Nemesis symbiotic activity (see Fig. 30B, top left and top right respectively). However transfer of the VIM-1 and the CTX-M-15 genes was found to be strongly inhibited in matings with recipients carrying spacers targeted against these genes: pNB104A and pNB104B (see plates 2A and 4B, bottom left and right respectively). In another experiment, an equivalent mating was done to test Nemesis symbiotic activity in recipients carrying all 8 spacer sequences in the plasmid pNB108. Here cells were picked directly from the mating mixture and streaked onto LBAp100Cm35 plates. Figure 30C plate, top left, again shows successful mating of donors 2, 4 and 5 with the recipient carrying pNB100 (see 2-, 4- and 5- in Fig. 30C). In all cases, however, mating with the recipient carrying pNB108 (see Fig. 30C, plate top right, 2/8, 4/8 and 5/8) gives strong inhibition of transfer of ampicillin resistance gene. And for comparison to a positive control in this assay the bottom plate with 5+ (see Fig. 30C) shows strong inhibition of transfer of TEM-1 ampicillin resistance gene to the recipient carrying pNB102.

The experiments reported above provide the proof-of-concept that, in the model organism, Escherichia coli, DNA constructs carrying the Cas9 CRISPR region plus a spacer region with sequences directed against a target region of the beta-lactamase gene can inactivate ampicillin resistance when delivered by naked DNA transformation and bacteriophage infection as well as prevent transfer of ampicillin resistance by plasmid conjugation. It is apparent that Nemesis symbiotics of the invention can be applied to pathogenic bacteria and for other antibiotic resistance genes.

Although the present invention has been described with reference to preferred or exemplary embodiments, those skilled in the art will recognise that various modifications and variations to the same can be accomplished without departing from the spirit and scope of the present invention and that such modifications are clearly contemplated herein. No limitation with respect to the specific embodiments disclosed herein and set forth in the appended claims is intended nor should any be inferred.

All documents cited herein are incorporated by reference in their entirety.

SEQUENCE LISTING

<110> Nemesis Bioscience Ltd

<120> Therapeutic

<130> P/71337.WO01/MAR

<150> GB1406674.0

<151> 2014-04-14

<150> GB1413719.4

<151> 2014-08-01

<150> GB1418508.6

<151> 2014-10-17

<160> 172

<170> Patentln version 3.5

<210> 1

<211> 12

<212> DNA

<213> Unknown

<220>

<223> CR05 bla gene sequence

<400> 1

gatacgggag gg

<210> 2

<211> 12

<212> DNA

<213> Unknown

<220>

<223> CR05 bla gene reverse strand

<400> 2

ccctcccgta tc 12

<210> 3

<211> 10

<212> DNA

<213> Unknown

<220>

<223> CR05 short bacterial off-target sequence

<400> 3

cgatacggga 10 <210> 4

<211> 11

<212> DNA

<213> Unknown

<220>

<223> CR05 short bacterial off-target sequence

<400> 4

cgatacggga g

<210> 5

<211> 11

<212> DNA

<213> Unknown

<220>

<223> CR05 short bacterial off-target sequence

<400> 5

gatacgggag g

<210> 6

<211> 19

<212> DNA

<213> Unknown

<220>

<223> CR30 bla gene sequence

<400> 6

tgctcatcat tggaaaacg

<210> 7

<211> 19

<212> DNA

<213> Unknown

<220>

<223> CR30 bla gene sequence reverse strand

<400> 7

cgttttccaa tgatgagca

<210> 8

<211> 12

<212> DNA

<213> Unknown

<220>

<223> CR30 short bacterial off-target sequence <400> 8

tcattggaaa ac 12

<210> 9

<211> 10

<212> DNA

<213> Unknown

<220>

<223> CR30 short bacterial off-target sequence

<400> 9

tgctcatcat

<210> 10

<211> 11

<212> DNA

<213> Unknown

CR30 short bacterial off-target sequence

<400> 10

gctcatcatt g 11

<210> 11

<211> 13

<212> DNA

<213> Unknown

<220>

<223> CR30 short bacterial off-target sequence

<400> 11

ctcatcattg gaa

<210> 12

<211> 17

<212> DNA

<213> Unknown

CR30 short bacterial off-target sequence

<400> 12

ctcagcattg caaaacg 17

<210> 13

<211> 15

<212> DNA

84

SUBSTITUTE SHEET RULE 26 <213> Unknown

<220>

<223> CR30 short bacterial off-target sequence

<400> 13

tcatcattga aaaac

<210> 14

<211> 12

<212> DNA

<213> Unknown

CR30 short bacterial off-target sequence

<400> 14

tcattggaaa ac

<210> 15

<211> 19

<212> DNA

<213> Unknown

<220>

<223> CR70 bla gene sequence

<400> 15

tcgccagtta atagtttgc

<210> 16

<211> 19

<212> DNA

<213> Unknown

<220>

<223> CR70 bla sequence reverse strand

<400> 16

agcggtcaat tatcaaacg

<210> 17

<211> 11

<212> DNA

<213> Unknown

<220>

<223> CR30 short bacterial off-target sequence - 1

<400> 17

tcgccagtta a <210> 18

<211> 12

<212> DNA

<213> Unknown

<220>

<223> CR70 short bacterial off-target sequence

<400> 18

tcgccagtta

<210> 19

<211> 11

<212> DNA

<213> Unknown

<220>

<223> CR70 short bacterial off-target sequence

<400> 19

cgccagttaa t

<210> 20

<211> 12

<212> DNA

<213> Unknown

<220>

<223> CR70 short bacterial off-target sequence

<400> 20

cgccagttaa ta

<210> 21

<211> 15

<212> DNA

<213> Unknown

<220>

<223> CR70 short bacterial off-target sequence

<400> 21

cgccagctaa tagtt 15

<210> 22

<211> 11

<212> DNA

<213> Unknown

<220>

<223> CR70 short bacterial off-target sequence <400> 22

ccagttaata g

<210> 23

<211> 11

<212> DNA

<213> Unknown

<220>

<223> CR70 short bacterial off-target sequence

<400> 23

taatagtttg c 11

<210> 24

<211> 20

<212> DNA

<213> Unknown

<220>

<223> CR90 bla sequence

<400> 24

ccgcgccaca tagcagaact

<210> 25

<211> 20

<212> DNA

<213> Unknown

<220>

<223> CR90 bla sequence reverse strand

<400> 25

agttctgcta tgtggcgcgg 20

<210> 26

<211> 11

<212> DNA

<213> Unknown

<220>

<223> CR90 short bacterial off-target sequence

<400> 26

acatagcaga a 11

<210> 27

<211> 11

<212> DNA <213> Unknown

<220>

<223> CR90 short bacterial off-target sequence

<400> 27

cgcgccacat a

<210> 28

<211> 13

<212> DNA

<213> Unknown

<220>

<223> CR90 short bacterial off-target sequence

<400> 28

cgcgccacat age

<210> 29

<211> 11

<212> DNA

<213> Unknown

CR90 short bacterial off-target sequence

<400> 29

gcgccacata g

<210> 30

<211> 11

<212> DNA

<213> Unknown

<220>

<223> CR90 short bacterial off-target sequence

<400> 30

gccacatagc a

<210> 31

<211> 11

<212> DNA

<213> Unknown

<220>

<223> CR90 short bacterial off-target sequence

<400> 31

acatagcaga a <210> 32

<211> 11

<212> DNA

<213> Unknown

CR90 short bacterial off-target sequence

<400> 32

catagcagaa c

<210> 33

<211> 16

<212> DNA

<213> Unknown

CR90 short bacterial off-target sequence

<400> 33

gcgccatata gcagaa

<210> 34

<211> 20

<212> RNA

<213> Artificial Sequence

Artificial RNA sequence

<220>

<221> stem_loop

<222> (1) .. (10)

<400> 34

uagauaacua cgauacggga

<210> 35

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Artificial RNA sequence

<220>

<221> stem_loop

<222> (1) .. (11)

<400> 35 acuuuaaaag ugcucaucau 20

<210> 36

<211> 20

<212> KNA

<213> Artificial sequence

Artificial RNA sequence

<220>

<221> stem_loop

<222> (3) .. (13)

<400> 36

acguugcgca aacuauuaac

<210> 37

<211> 20

<212> KNA

<213> Artificial sequence

Artificial RNA sequence

<220>

<221> stem_loop

<222> (9) .. (17)

<400> 37

acuuuuaaag uucugcuaug

<210> 38

<211> 861

<212> DNA

<213> Unknown

<220>

<223> Beta-lactamase gene from pBR322

<400> 38

atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840 tcactgatta agcattggta a 861

<210> 39

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence

<400> 39

ccgcgtaggc atgatagaaa

<210> 40

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence

<400> 40

acgttaaaca ccgccattcc

<210> 41

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence

<400> 41

gcgctggaga aaagcagcgg 20

91

SUBSTITUTE SHEET RULE 26 <210> 42

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 42

aagctgattg cccatctggg

<210> 43

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 43

acgctcaaca ccgcgatccc

<210> 44

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 44

aactacttac tctagcttcc

<210> 45

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 45

ttgttgctga aggagttggg

<210> 46

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 46

agcgaaaaac accttgccga 20

<210> 47

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence

<400> 47

ctgggaaacg gcactgaatg

<210> 48

<211> 20

<212> DNA

<213> Unknown

Spacer sequence

<400> 48

tgggttgttg gagagaaaac 20

<210> 49

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence

<400> 49

aaacacagcg gcacttctcg

<210> 50

<211> 20

<212> DNA

<213> Unknown

Spacer sequence

<400> 50

aaaattgaag ttttttatcc 20

<210> 51

<211> 20

<212> DNA <213> Unknown

<220>

<223> Spacer sequence <400> 51

tggcagccgc agtggaagcc

<210> 52

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 52

tcacagctac ttgaaggttc

<210> 53

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 53

atcaaaactg gcagccgcaa

<210> 54

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence

<400> 54

atcaaaactg gcagccgcaa

<210> 55

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 55

cagtactcca accccagcat <210> 56

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 56

tcacctggcc gcaaatagtc

<210> 57

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 57

acaacggatt aacagaagca

<210> 58

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 58

agaacatcag cgcttggtca

<210> 59

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 59

ataacggctt gacccagtca

<210> 60

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 60

ggcaaccaga atatcagtgg 20

<210> 61

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence

<400> 61

ggatgccggt gacgaacagc

<210> 62

<211> 20

<212> DNA

<213> Unknown

Spacer sequence

<400> 62

gctacagtac agcgataacg 20

<210> 63

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence

<400> 63

gacgttgcgt cagcttacgc

<210> 64

<211> 20

<212> DNA

<213> Unknown

Spacer sequence

<400> 64

aactacttac tctagcttcc 20

<210> 65

<211> 20

<212> DNA <213> Unknown

<220>

<223> Spacer sequence <400> 65

ttgttgctga aggagttggg

<210> 66

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 66

agcgaaaaac accttgccga

<210> 67

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 67

acctttaaag tgctgctgtg

<210> 68

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 68

tgggttgttg gagagaaaac

<210> 69

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 69

aaacacagcg gcacttctcg <210> 70

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 70

aaaattgaag ttttttatcc

<210> 71

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 71

ggtttgatcg tcagggatgg

<210> 72

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 72

gtggattaac gttccgaaag

<210> 73

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 73

cagcgacagc aaagtggcat

<210> 74

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 74

cttgccacct acagtgcggg

<210> 75

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 75

cccccaaagg aatggagatc

<210> 76

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 76

caccaagtct ttaagtggga

<210> 77

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 77

ataacggctt gacccagtca

<210> 78

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence <400> 78

ctgggaaacg gaactgaatg

<210> 79

<211> 20

<212> DNA <213> Unknown

<220>

<223> Spacer sequence

<400> 79

acgttaaaca ccgccattcc

<210> 80

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence

<400> 80

aactacttac tctagcttcc

<210> 81

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence

<400> 81

ttgttgctga aggagttggg

<210> 82

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence

<400> 82

aaacacagcg gcacttctcg

<210> 83

<211> 34

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence

<400> 83

ggctagttaa aaataaaatt gaagtttttt 34 <210> 84

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence

<400> 84

ggtttgatcg tcagggatgg

<210> 85

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Spacer sequence

<400> 85

ataacggctt gacccagtca

<210> 86

<211> 195

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 86

ccaaaactga gacctgctgc ggacgtccaa aggtctcgtt ttagagctat gctgttttga 60 atggtcccaa aacttgccga tgataacttg agaaagaggg ttaataccag cagtcggata 120 ccttcctatt ctttctgtta aagcgttttc atgttataat aggcaaattt tagatgaaga 180 ttatttctta ataac 195

<210> 87

<211> 81

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 87

tctaaaacga agagcgctgc gtccgagacg ctcttcagtt ttgggaccat tcaaaacagc 60 atagctctaa aacctcgtag a 81 <210> 88

<211> 54

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 88

gggctggcaa gccacgtttg gtgggtctcg aaacggtttg atcgtcaggg atgg 54

<210> 89

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 89

ggataaaaaa cttcaatttt atttttaact agccgttttg ggaccattca aaacagcata gctctaaaac ccatccctga cgatcaaacc

<210> 90

<211> 90

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 90

ggctagttaa aaataaaatt gaagtttttt atccgtttta gagctatgct gttttgaatg gtcccaaaac aaacacagcg gcacttctcg

<210> 91

<211> 76

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 91

cccaactcct tcagcaacaa gttttgggac cattcaaaac agcatagctc taaaaccgag 60 aagtgccgct gtgttt 76

<210> 92 <211> 76

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 92

ttgttgctga aggagttggg gttttagagc tatgctgttt tgaatggtcc caaaacgtcc 60 atcccactta aagact 76

<210> 93

<211> 76

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 93

cattcagttc cgtttcccag gttttgggac cattcaaaac agcatagctc taaaacagtc tttaagtggg atggac

<210> 94

<211> 76

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 94

ctgggaaacg gaactgaatg gttttagagc tatgctgttt tgaatggtcc caaaacaact acttactcta gcttcc

<210> 95

<211> 111

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 95

ccgggagctg catgtgtcag aggggtctcc aaaacggaat ggcggtgttt aacgtgtttt 60 gggaccattc aaaacagcat agctctaaaa cggaagctag agtaagtagt t 111

<210> 96 <211> 111

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 96

ccgggagctg catgtgtcag aggggtctcc aaaaccccaa ctccttcagc aacaagtttt gggaccattc aaaacagcat agctctaaaa ccgagaagtg ccgctgtgtt t

<210> 97

<211> 54

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 97

gggctggcaa gccacgtttg gtggctcttc aaacgtccat cccacttaaa gact

<210> 98

<211> 111

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 98

ccgggagctg catgtgtcag agggctcttc aaaacggaat ggcggtgttt aacgtgtttt gggaccattc aaaacagcat agctctaaaa cggaagctag agtaagtagt t

<210> 99

<211> 1368

<212> PRT

<213> Streptococcus pyogenes Ml GAS

<400> 99

Met Asp Lys Lys Tyr Ser lie Gly Leu Asp lie Gly Thr Asn Ser Val 1 5 10 15

Gly Trp Ala Val lie Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser lie Lys Lys Asn Leu lie

35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg lie Cys 65 70 75 80

Tyr Leu Gin Glu lie Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro lie Phe Gly Asn lie Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr lie Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu lie Tyr Leu Ala Leu Ala His 145 150 155 160

Met lie Lys Phe Arg Gly His Phe Leu lie Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe lie Gin Leu Val Gin Thr Tyr

180 185 190

Asn Gin Leu Phe Glu Glu Asn Pro lie Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala lie Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220

Leu lie Ala Gin Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240

Leu lie Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gin Leu Ser Lys Asp Thr Tyr Asp

260 265 270

105

SUBSTITUTE SHEET RULE 26 Asp Asp Leu Asp Asn Leu Leu Ala Gin lie Gly Asp Gin Tyr Ala Asp 275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala lie Leu Leu Ser Asp 290 295 300 lie Leu Arg Val Asn Thr Glu lie Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320

Met lie Lys Arg Tyr Asp Glu His His Gin Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gin Gin Leu Pro Glu Lys Tyr Lys Glu lie Phe Phe

340 345 350

Asp Gin Ser Lys Asn Gly Tyr Ala Gly Tyr lie Asp Gly Gly Ala Ser

355 360 365

Gin Glu Glu Phe Tyr Lys Phe lie Lys Pro lie Leu Glu Lys Met Asp 370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400

Lys Gin Arg Thr Phe Asp Asn Gly Ser lie Pro His Gin lie His Leu

405 410 415

Gly Glu Leu His Ala lie Leu Arg Arg Gin Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys lie Glu Lys lie Leu Thr Phe Arg lie

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460

Met Thr Arg Lys Ser Glu Glu Thr lie Thr Pro Trp Asn Phe Glu Glu 465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gin Ser Phe lie Glu Arg Met Thr

485 490 495

106

SUBSTITUTE SHEET RULE 26 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gin 530 535 540

Lys Lys Ala lie Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560

Val Lys Gin Leu Lys Glu Asp Tyr Phe Lys Lys lie Glu Cys Phe Asp

565 570 575

Ser Val Glu lie Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys lie lie Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp lie Leu Glu Asp lie Val Leu Thr Leu Thr 610 615 620

Leu Phe Glu Asp Arg Glu Met lie Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gin Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu lie Asn Gly lie Arg Asp

660 665 670

Lys Gin Ser Gly Lys Thr lie Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gin Leu lie His Asp Asp Ser Leu Thr Phe 690 695 700

Lys Glu Asp lie Gin Lys Ala Gin Val Ser Gly Gin Gly Asp Ser Leu 705 710 715 720

107

SUBSTITUTE SHEET RULE 26 His Glu His lie Ala Asn Leu Ala Gly Ser Pro Ala lie Lys Lys Gly 725 730 735 lie Leu Gin Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn lie Val lie Glu Met Ala Arg Glu Asn Gin

755 760 765

Thr Thr Gin Lys Gly Gin Lys Asn Ser Arg Glu Arg Met Lys Arg lie 770 775 780

Glu Glu Gly lie Lys Glu Leu Gly Ser Gin lie Leu Lys Glu His Pro 785 790 795 800

Val Glu Asn Thr Gin Leu Gin Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gin Asn Gly Arg Asp Met Tyr Val Asp Gin Glu Leu Asp lie Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His lie Val Pro Gin Ser Phe Leu Lys

835 840 845

Asp Asp Ser lie Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880

Asn Tyr Trp Arg Gin Leu Leu Asn Ala Lys Leu lie Thr Gin Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe lie Lys Arg Gin Leu Val Glu Thr Arg Gin lie Thr

915 920 925

Lys His Val Ala Gin lie Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940

108

SUBSTITUTE SHEET RULE 26 Glu Asn Asp Lys Leu lie Arg Glu Val Lys Val lie Thr Leu Lys Ser 945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gin Phe Tyr Lys Val Arg

965 970 975

Glu lie Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu lie Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met lie Ala 1010 1015 1020

Lys Ser Glu Gin Glu lie Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035

Tyr Ser Asn lie Met Asn Phe Phe Lys Thr Glu lie Thr Leu Ala 1040 1045 1050

Asn Gly Glu lie Arg Lys Arg Pro Leu lie Glu Thr Asn Gly Glu 1055 1060 1065

Thr Gly Glu lie Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080

Arg Lys Val Leu Ser Met Pro Gin Val Asn lie Val Lys Lys Thr 1085 1090 1095

Glu Val Gin Thr Gly Gly Phe Ser Lys Glu Ser lie Leu Pro Lys 1100 1105 1110

Arg Asn Ser Asp Lys Leu lie Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155

109

SUBSTITUTE SHEET RULE 26 Ser Val Lys Glu Leu Leu Gly lie Thr lie Met Glu Arg Ser Ser 1160 1165 1170

Phe Glu Lys Asn Pro lie Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185

Glu Val Lys Lys Asp Leu lie lie Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200

Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215

Glu Leu Gin Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245

Pro Glu Asp Asn Glu Gin Lys Gin Leu Phe Val Glu Gin His Lys 1250 1255 1260

His Tyr Leu Asp Glu lie lie Glu Gin lie Ser Glu Phe Ser Lys 1265 1270 1275

Arg Val lie Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290

Tyr Asn Lys His Arg Asp Lys Pro lie Arg Glu Gin Ala Glu Asn 1295 1300 1305 lie lie His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320

Phe Lys Tyr Phe Asp Thr Thr lie Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335

Thr Lys Glu Val Leu Asp Ala Thr Leu lie His Gin Ser lie Thr 1340 1345 1350

Gly Leu Tyr Glu Thr Arg lie Asp Leu Ser Gin Leu Gly Gly Asp 1355 1360 1365

110

SUBSTITUTE SHEET RULE 26 <210> 100

<211> 20

<212> DNA

<213> Unknown

Anti-protospacer sequence

<400> 100

tagataacta cgatacggga

<210> 101

<211> 20

<212> DNA

<213> Unknown

Anti-protospacer sequence

<400> 101

gatcgttgtc agaagt;

<210> 102

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Anti-protospacer sequence

<400> 102

actttaaaag tgctcatcat

<210> 103

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Anti-protospacer sequence

<400> 103

tttactttca ccagcgtttc 20

<210> 104

<211> 20

<212> DNA

<213> Unknown

<220> <223> Anti-protospacer sequence <400> 104

attaatagac tggatggagg

<210> 105

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Anti-protospacer sequence

<400> 105

acaattaata gactggatgg

<210> 106

<211> 20

<212> DNA

<213> Unknown

Anti-protospacer sequence

<400> 106

gcaacaatta atagactgga

<210> 107

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Anti-protospacer sequence <400> 107

cccggcaaca attaatagac

<210> 108

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Anti-protospacer sequence

<400> 108

aactacttac tctagcttcc

<210> 109

<211> 20 <212> DNA

<213> Unknown

<220>

<223> Anti-protospacer sequence

<400> 109

acgttgcgca aactattaac 20

<210> 110

<211> 20

<212> DNA

<213> Unknown

Anti-protospacer sequence

<400> 110

tgtaactcgc cttgatcgtt 20

<210> 111

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Anti-protospacer sequence

<400> 111

atgtaactcg ccttgatcgt 20

<210> 112

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Anti-protospacer sequence

<400> 112

aacttacttc tgacaacgat 20

<210> 113

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Anti-protospacer sequence

<400> 113

agtcacagaa aagcatctta 20 <210> 114

<211> 20

<212> DNA

<213> Unknown

<220>

<223> Anti-protospacer sequence

<400> 114

acttttaaag ttctgctatg

<210> 115

<211> 25

<212> DNA

<213> Unknown

<220>

<223> Adaptor sequence

<400> 115

aaactagata actacgatac gggag

<210> 116

<211> 25

<212> DNA

<213> Unknown

<220>

<223> Adaptor sequence

<400> 116

aaaactcccg tatcgtagtt atcta

<210> 117

<211> 25

<212> DNA

<213> Unknown

<220>

<223> Adaptor sequence

<400> 117

aaacacttta aaagtgctca tcatg 25

<210> 118

<211> 25

<212> DNA

<213> Unknown

<220> <223> Adaptor sequence

<400> 118

aaaacatgat gagcactttt aa

<210> 119

<211> 25

<212> DNA

<213> Unknown

<220>

<223> Adaptor sequence

<400> 119

aaacacgttg cgcaaactat taacg 25

<210> 120

<211> 25

<212> DNA

<213> Unknown

<220>

<223> Adaptor sequence

<400> 120

aaaacgttaa tagtttgcgc aa

<210> 121

<211> 25

<212> DNA

<213> Unknown

<220>

<223> Adaptor sequence

<400> 121

aaacactttt aaagttctgc tatgg 25

<210> 122

<211> 25

<212> DNA

<213> Unknown

<220>

<223> Adaptor sequence

<400> 122

aaaaccatag cagaacttta aa

<210> 123

<211> 81 <212> DNA

<213> Unknown

<220>

<223> Adaptor sequence for dual targets

<400> 123

aaacacttta aaagtgctca tcatgtttta gagctatgct gttttgaatg gtcccaaaac 60 acttttaaag ttctgctatg g 81

<210> 124

<211> 81

<212> DNA

<213> Unknown

<220>

<223> Adaptor sequence for dual targets

<400> 124

aaaaccatag cagaacttta aaagtgtttt gggaccattc aaaacagcat agctctaaaa 60 catgatgagc acttttaaag t 81

<210> 125

<211> 9326

<212> DNA

<213> Artificial

<220>

<223> Plasmid sequence

<400> 125

gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt 60 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt 120 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga 180 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga 240 aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt 300 ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc 360 ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat 420 ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt 480 gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg 540 acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg agtgtatact 600 ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa 660

116

SUBSTITUTE SHEET RULE 26 aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc 720 actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc 780 ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa 840 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc 900 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc 960 tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc 1020 gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac 1080 tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt 1140 gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt 1200 agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg 1260 tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt 1320 cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc 1380 aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga tttcagtgca 1440 atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctc 1500 atgtttgaca gcttatcatc gataagcttt aatgcggtag tttatcacag ttaaattgct 1560 aacgcagtca ggcaccgtgt atgaaatcta acaatgcgct catcgtcatc ctcggcaccg 1620 tcaccctgga tgctgtaggc ataggcttgg ttatgccggt actgccgggc ctcttgcggg 1680 attacgaaat catcctgtgg agcttagtag gtttagcaag atggcagcgc ctaaatgtag 1740 aatgataaaa ggattaagag attaatttcc ctaaaaatga taaaacaagc gttttgaaag 1800 cgcttgtttt tttggtttgc agtcagagta gaatagaagt atcaaaaaaa gcaccgactc 1860 ggtgccactt tttcaagttg ataacggact agccttattt taacttgcta tgctgttttg 1920 aatggttcca acaagattat tttataactt ttataacaaa taatcaagga gaaattcaaa 1980 gaaatttatc agccataaaa caatacttaa tactatagaa tgataacaaa ataaactact 2040 ttttaaaaga attttgtgtt ataatctatt tattattaag tattgggtaa tattttttga 2100 agagatattt tgaaaaagaa aaattaaagc atattaaact aatttcggag gtcattaaaa 2160 ctattattga aatcatcaaa ctcattatgg atttaattta aactttttat tttaggaggc 2220 aaaaatggat aagaaatact caataggctt agatatcggc acaaatagcg tcggatgggc 2280 ggtgatcact gatgaatata aggttccgtc taaaaagttc aaggttctgg gaaatacaga 2340

117

SUBSTITUTE SHEET RULE 26 ccgccacagt atcaaaaaaa atcttatagg ggctctttta tttgacagtg gagagacagc 2400 ggaagcgact cgtctcaaac ggacagctcg tagaaggtat acacgtcgga agaatcgtat 2460 ttgttatcta caggagattt tttcaaatga gatggcgaaa gtagatgata gtttctttca 2520 tcgacttgaa gagtcttttt tggtggaaga agacaagaag catgaacgtc atcctatttt 2580 tggaaatata gtagatgaag ttgcttatca tgagaaatat ccaactatct atcatctgcg 2640 aaaaaaattg gtagattcta ctgataaagc ggatttgcgc ttaatctatt tggccttagc 2700 gcatatgatt aagtttcgtg gtcatttttt gattgaggga gatttaaatc ctgataatag 2760 tgatgtggac aaactattta tccagttggt acaaacctac aatcaattat ttgaagaaaa 2820 ccctattaac gcaagtggag tagatgctaa agcgattctt tctgcacgat tgagtaaatc 2880 aagacgatta gaaaatctca ttgctcagct ccccggtgag aagaaaaatg gcttatttgg 2940 gaatctcatt gctttgtcat tgggtttgac ccctaatttt aaatcaaatt ttgatttggc 3000 agaagatgct aaattacagc tttcaaaaga tacttacgat gatgatttag ataatttatt 3060 ggcgcaaatt ggagatcaat atgctgattt gtttttggca gctaagaatt tatcagatgc 3120 tattttactt tcagatatcc taagagtaaa tactgaaata actaaggctc ccctatcagc 3180 ttcaatgatt aaacgctacg atgaacatca tcaagacttg actcttttaa aagctttagt 3240 tcgacaacaa cttccagaaa agtataaaga aatctttttt gatcaatcaa aaaacggata 3300 tgcaggttat attgatgggg gagctagcca agaagaattt tataaattta tcaaaccaat 3360 tttagaaaaa atggatggta ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct 3420 gcgcaagcaa cggacctttg acaacggctc tattccccat caaattcact tgggtgagct 3480 gcatgctatt ttgagaagac aagaagactt ttatccattt ttaaaagaca atcgtgagaa 3540 gattgaaaaa atcttgactt ttcgaattcc ttattatgtt ggtccattgg cgcgtggcaa 3600 tagtcgtttt gcatggatga ctcggaagtc tgaagaaaca attaccccat ggaattttga 3660 agaagttgtc gataaaggtg cttcagctca atcatttatt gaacgcatga caaactttga 3720 taaaaatctt ccaaatgaaa aagtactacc aaaacatagt ttgctttatg agtattttac 3780 ggtttataac gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa aaccagcatt 3840 tctttcaggt gaacagaaga aagccattgt tgatttactc ttcaaaacaa atcgaaaagt 3900 aaccgttaag caattaaaag aagattattt caaaaaaata gaatgttttg atagtgttga 3960 aatttcagga gttgaagata gatttaatgc ttcattaggt acctaccatg atttgctaaa 4020

118

SUBSTITUTE SHEET RULE 26 aattattaaa gataaagatt ttttggataa tgaagaaaat gaagatatct tagaggatat 4080 tgttttaaca ttgaccttat ttgaagatag ggagatgatt gaggaaagac ttaaaacata 4140 tgctcacctc tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt atactggttg 4200 gggacgtttg tctcgaaaat tgattaatgg tattagggat aagcaatctg gcaaaacaat 4260 attagatttt ttgaaatcag atggttttgc caatcgcaat tttatgcagc tgatccatga 4320 tgatagtttg acatttaaag aagacattca aaaagcacaa gtgtctggac aaggcgatag 4380 tttacatgaa catattgcaa atttagctgg tagccctgct attaaaaaag gtattttaca 4440 gactgtaaaa gttgttgatg aattggtcaa agtaatgggg cggcataagc cagaaaatat 4500 cgttattgaa atggcacgtg aaaatcagac aactcaaaag ggccagaaaa attcgcgaga 4560 gcgtatgaaa cgaatcgaag aaggtatcaa agaattagga agtcagattc ttaaagagca 4620 tcctgttgaa aatactcaat tgcaaaatga aaagctctat ctctattatc tccaaaatgg 4680 aagagacatg tatgtggacc aagaattaga tattaatcgt ttaagtgatt atgatgtcga 4740 tcacattgtt ccacaaagtt tccttaaaga cgattcaata gacaataagg tcttaacgcg 4800 ttctgataaa aatcgtggta aatcggataa cgttccaagt gaagaagtag tcaaaaagat 4860 gaaaaactat tggagacaac ttctaaacgc caagttaatc actcaacgta agtttgataa 4920 tttaacgaaa gctgaacgtg gaggtttgag tgaacttgat aaagctggtt ttatcaaacg 4980 ccaattggtt gaaactcgcc aaatcactaa gcatgtggca caaattttgg atagtcgcat 5040 gaatactaaa tacgatgaaa atgataaact tattcgagag gttaaagtga ttaccttaaa 5100 atctaaatta gtttctgact tccgaaaaga tttccaattc tataaagtac gtgagattaa 5160 caattaccat catgcccatg atgcgtatct aaatgccgtc gttggaactg ctttgattaa 5220 gaaatatcca aaacttgaat cggagtttgt ctatggtgat tataaagttt atgatgttcg 5280 taaaatgatt gctaagtctg agcaagaaat aggcaaagca accgcaaaat atttctttta 5340 ctctaatatc atgaacttct tcaaaacaga aattacactt gcaaatggag agattcgcaa 5400 acgccctcta atcgaaacta atggggaaac tggagaaatt gtctgggata aagggcgaga 5460 ttttgccaca gtgcgcaaag tattgtccat gccccaagtc aatattgtca agaaaacaga 5520 agtacagaca ggcggattct ccaaggagtc aattttacca aaaagaaatt cggacaagct 5580 tattgctcgt aaaaaagact gggatccaaa aaaatatggt ggttttgata gtccaacggt 5640 agcttattca gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga agttaaaatc 5700

119

SUBSTITUTE SHEET RULE 26 cgttaaagag ttactaggga tcacaattat ggaaagaagt tcctttgaaa aaaatccgat 5760 tgacttttta gaagctaaag gatataagga agttaaaaaa gacttaatca ttaaactacc 5820 taaatatagt ctttttgagt tagaaaacgg tcgtaaacgg atgctggcta gtgccggaga 5880 attacaaaaa ggaaatgagc tggctctgcc aagcaaatat gtgaattttt tatatttagc 5940 tagtcattat gaaaagttga agggtagtcc agaagataac gaacaaaaac aattgtttgt 6000 ggagcagcat aagcattatt tagatgagat tattgagcaa atcagtgaat tttctaagcg 6060 tgttatttta gcagatgcca atttagataa agttcttagt gcatataaca aacatagaga 6120 caaaccaata cgtgaacaag cagaaaatat tattcattta tttacgttga cgaatcttgg 6180 agctcccgct gcttttaaat attttgatac aacaattgat cgtaaacgat atacgtctac 6240 aaaagaagtt ttagatgcca ctcttatcca tcaatccatc actggtcttt atgaaacacg 6300 cattgatttg agtcagctag gaggtgactg aagtatattt tagatgaaga ttatttctta 6360 ataactaaaa atatggtata atactcttaa taaatgcagt aatacagggg cttttcaaga 6420 ctgaagtcta gctgagacaa atagtgcgat tacgaaattt tttagacaaa aatagtctac 6480 gaggttttag agctatgctg ttttgaatgg tcccaaaact gagaccagtc tcggaagctc 6540 aaaggtctcg ttttagagct atgctgtttt gaatggtccc aaaacttcag cacactgaga 6600 cttgttgagt tccatgtttt agagctatgc tgttttgaat ggactccatt caacattgcc 6660 gatgataact tgagaaagag ggttaatacc agcagtcgga taccttccta ttctttctgt 6720 taaagcgttt tcatgttata ataggcaaaa gaagagtagt gtgatcgtcc attccgacag 6780 catcgccagt cactatggcg tgctgctagc gctatatgcg ttgatgcaat ttctatgcgc 6840 acccgttctc ggagcactgt ccgaccgctt tggccgccgc ccagtcctgc tcgcttcgct 6900 acttggagcc actatcgact acgcgatcat ggcgaccaca cccgtcctgt ggatcctcta 6960 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat 7020 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt 7080 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca 7140 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct 7200 aatgcaggag tcgcataagg gagagcgtcg accgatgccc ttgagagcct tcaacccagt 7260 cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt 7320 tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg 7380

120

SUBSTITUTE SHEET RULE 26 ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc 7440 cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat 7500 tatcgccggc atggcggccg acgcgctggg ctacgtcttg ctggcgttcg cgacgcgagg 7560 ctggatggcc ttccccatta tgattcttct cgcttccggc ggcatcggga tgcccgcgtt 7620 gcaggccatg ctgtccaggc aggtagatga cgaccatcag ggacagcttc aaggatcgct 7680 cgcggctctt accagcctaa cttcgatcat tggaccgctg atcgtcacgg cgatttatgc 7740 cgcctcggcg agcacatgga acgggttggc atggattgta ggcgccgccc tataccttgt 7800 ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct gaatggaagc 7860 cggcggcacc tcgctaacgg attcaccact ccaagaattg gagccaatca attcttgcgg 7920 agaactgtga atgcgcaaac caacccttgg cagaacatat ccatcgcgtc cgccatctcc 7980 agcagccgca cgcggcgcat ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc 8040 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat 8100 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga 8160 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtcc 8220 cctacgtgct gctgaagttg cccgcaacag agagtggaac caaccggtga taccacgata 8280 ctatgactga gagtcaacgc catgagcggc ctcatttctt attctgagtt acaacagtcc 8340 gcaccgctgt ccggtagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt 8400 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgcc caacagtccc 8460 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgccctgcac cattatgttc 8520 cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg 8580 aagcgctaac cgtttttatc aggctctggg aggcagaata aatgatcata tcgtcaatta 8640 ttacctccac ggggagagcc tgagcaaact ggcctcaggc atttgagaag cacacggtca 8700 cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac 8760 gaccctgccc tgaaccgacg accgggtcga atttgctttc gaatttctgc cattcatccg 8820 cttattatca cttattcagg cgtagcacca ggcgtttaag ggcaccaata actgccttaa 8880 aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg 8940 ccgacatgga agccatcaca gacggcatga tgaacctgaa tcgccagcgg catcagcacc 9000 ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata 9060

121

SUBSTITUTE SHEET RULE 26 ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac 9120 atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct 9180 tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa 9240 aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc 9300 agctcaccgt ctttcattgc catacg 9326

<210> 126

<211> 4107

<212> DNA

<213> Artificial

<220>

<223> Cas9 coding sequence

<400> 126

atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg 60 atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120 cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa 180 gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240 tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300 cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360 aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420 aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480 atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540 gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct 600 attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660 cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat 720 ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa 780 gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg 840 caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900 ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca 960 atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020 caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080

122

SUBSTITUTE SHEET RULE 26 ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140 gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200 aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260 gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320 gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380 cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440 gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500 aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560 tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt 1620 tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1680 gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740 tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt 1800 attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860 ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct 1920 cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980 cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040 gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100 agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2160 catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220 gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280 attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt 2340 atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct 2400 gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga 2460 gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac 2520 attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct 2580 gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa 2640 aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta 2700 acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa 2760

123

SUBSTITUTE SHEET RULE 26 ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat 2820 actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct 2880 aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat 2940 taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa 3000 tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa 3060 atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct 3120 aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc 3180 cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt 3240 gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3300 cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt 3360 gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct 3420 tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt 3480 aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3540 tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa 3600 tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta 3660 caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt 3720 cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag 3780 cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt 3840 attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa 3900 ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3960 cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4020 gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4080 gatttgagtc agctaggagg tgactga 4107

<210> 127

<211> 69

<212> DNA

<213> Artificial

<220>

<223> -10 promoter in pCas9

124

SUBSTITUTE SHEET RULE 26 <400> 127

agtatatttt agatgaagat tatttottaa taactaaaaa tatggtataa tactcttaat 60 aaatgcagt 69

<210> 128

<211> 80

<212> DNA

<213> Artificial

<220>

<223> Leader sequence in pCas9

<400> 128

aatacagggg cttttcaaga ctgaagtcta gctgagacaa atagtgcgat tacgaaattt 60 tttagacaaa aatagtctac 80

<210> 129

<211> 194

<212> DNA

<213> Artificial

<220>

<223> Spacer sequence

<400> 129

aaaactgaga ccgggcagtg agcgcaacgc aattaacatt aggcacccca ggcttgacaa 60 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacggaggt 120 cacatatgag ataataataa ctagctgaat tccccagccc gcctaatgag cgggcttttt 180 tttggtctcg tttt 194

<210> 130

<211> 86

<212> RNA

<213> Artificial

<220>

<223> tracrRNA

<400> 130

ggaaccauuc aaaacagcau agcaaguuaa aauaaggcua guccguuauc aacuugaaaa 60 aguggcaccg agucggugcu uuuuuu 86

<210> 131

<211> 91

<212> DNA

<213> Artificial <220>

<223> pre-crRNA

<400> 131

guuu agagc uaugctguuu ugaauggucc caaacacuuu uaaaguucug cuaugguuuu 60 agagcuaugc tguuuugaau ggucccaaaa c 91

<210> 132

<211> 11

<212> RNA

<213> Artificial

<220>

<223> Cleaved tracrRNA after first processing event

<400> 132

ggaaccauuc a 11

<210> 133

<211> 75

<212> DNA

<213> Artificial

<220>

<223> Cleaved tracrRNA

<400> 133

aaacagcaua gcaaguuaaa auaaggcuag uccguuauca acuugaaaaa guggcaccga gucggugcuu uuuuu

<210> 134

<211> 55

<212> DNA

<213> Artificial

<220>

<223> cleaved pre-crRNA

<400> 134

aaugguccca aacacuuuua aaguucugcu augguuuuag agcuaugctg uuuug

<210> 135

<211> 42

<212> DNA

<213> Artificial

<220>

<223> Cleaved pre-crRNA after 2nd processing event <400> 135

acuuuuaaag uucugcuaug guuuuagagc uaugctguuu ug

<210> 136

<211> 20

<212> DNA

<213> Unknown crRNA complementary sequence within target beta-lactamase DNA sequence

<400> 136

acttttaaag ttctgctatg 20

<210> 137

<211> 38

<212> DNA

<213> Unknown

CR90 -containing target beta-lactamase DNA sequence

anti-protospacer strand

<400> 137

ccaagtcaac ttttaaagtt ctgctatgtg gcgcggta 38

<210> 138

<211> 38

<212> DNA

<213> Unknown

<220>

<223> CR90 -containing target beta-lactamase DNA sequence protospacer strand

<400> 138

taccgcgcca catagcagaa ctttaaaagt tgacttgg 38

<210> 139

<211> 20

<212> DNA

<213> Unknown

<220>

<223> crRNA complementary sequence within target beta-lactamase DNA

sequence - reverse strand

<400> 139

catagcagaa ctttaaaagt 20 <210> 140

<211> 4758

<212> DNA

<213> Streptococcus pyogenes

<400> 140

atgccggtac tgccgggcct cttgcgggat ccagaagtct ttttcttgca ctgtttcctt 60 ttctttatga tagtttacga aatcatcctg tggagcttag taggtttagc aagatggcag 120 cgcctaaatg tagaatgata aaaggattaa gagattaatt tccctaaaaa tgataaaaca 180 agcgttttga aagcgcttgt ttttttggtt tgcagtcaga gtagaataga agtatcaaaa 240 aaagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg 300 ctatgctgtt ttgaatggtt ccaacaagat tattttataa cttttataac aaataatcaa 360 ggagaaattc aaagaaattt atcagccata aaacaatact taatactata gaatgataac 420 aaaataaact actttttaaa agaattttgt gttataatct atttattatt aagtattggg 480 taatattttt tgaagagata ttttgaaaaa gaaaaattaa agcatattaa actaatttcg 540 gaggtcatta aaactattat tgaaatcatc aaactcatta tggatttaat ttaaactttt 600 tattttagga ggcaaaaatg gataagaaat actcaatagg cttagatatc ggcacaaata 660 gcgtcggatg ggcggtgatc actgatgaat ataaggttcc gtctaaaaag ttcaaggttc 720 tgggaaatac agaccgccac agtatcaaaa aaaatcttat aggggctctt ttatttgaca 780 gtggagagac agcggaagcg actcgtctca aacggacagc tcgtagaagg tatacacgtc 840 ggaagaatcg tatttgttat ctacaggaga ttttttcaaa tgagatggcg aaagtagatg 900 atagtttctt tcatcgactt gaagagtctt ttttggtgga agaagacaag aagcatgaac 960 gtcatcctat ttttggaaat atagtagatg aagttgctta tcatgagaaa tatccaacta 1020 tctatcatct gcgaaaaaaa ttggtagatt ctactgataa agcggatttg cgcttaatct 1080 atttggcctt agcgcatatg attaagtttc gtggtcattt tttgattgag ggagatttaa 1140 atcctgataa tagtgatgtg gacaaactat ttatccagtt ggtacaaacc tacaatcaat 1200 tatttgaaga aaaccctatt aacgcaagtg gagtagatgc taaagcgatt ctttctgcac 1260 gattgagtaa atcaagacga ttagaaaatc tcattgctca gctccccggt gagaagaaaa 1320 atggcttatt tgggaatctc attgctttgt cattgggttt gacccctaat tttaaatcaa 1380 attttgattt ggcagaagat gctaaattac agctttcaaa agatacttac gatgatgatt 1440 tagataattt attggcgcaa attggagatc aatatgctga tttgtttttg gcagctaaga 1500

128

SUBSTITUTE SHEET RULE 26 atttatcaga tgctatttta ctttcagata tcctaagagt aaatactgaa ataactaagg 1560 ctcccctatc agcttcaatg attaaacgct acgatgaaca tcatcaagac ttgactcttt 1620 taaaagcttt agttcgacaa caacttccag aaaagtataa agaaatcttt tttgatcaat 1680 caaaaaacgg atatgcaggt tatattgatg ggggagctag ccaagaagaa ttttataaat 1740 ttatcaaacc aattttagaa aaaatggatg gtactgagga attattggtg aaactaaatc 1800 gtgaagattt gctgcgcaag caacggacct ttgacaacgg ctctattccc catcaaattc 1860 acttgggtga gctgcatgct attttgagaa gacaagaaga cttttatcca tttttaaaag 1920 acaatcgtga gaagattgaa aaaatcttga cttttcgaat tccttattat gttggtccat 1980 tggcgcgtgg caatagtcgt tttgcatgga tgactcggaa gtctgaagaa acaattaccc 2040 catggaattt tgaagaagtt gtcgataaag gtgcttcagc tcaatcattt attgaacgca 2100 tgacaaactt tgataaaaat cttccaaatg aaaaagtact accaaaacat agtttgcttt 2160 atgagtattt tacggtttat aacgaattga caaaggtcaa atatgttact gaaggaatgc 2220 gaaaaccagc atttctttca ggtgaacaga agaaagccat tgttgattta ctcttcaaaa 2280 caaatcgaaa agtaaccgtt aagcaattaa aagaagatta tttcaaaaaa atagaatgtt 2340 ttgatagtgt tgaaatttca ggagttgaag atagatttaa tgcttcatta ggtacctacc 2400 atgatttgct aaaaattatt aaagataaag attttttgga taatgaagaa aatgaagata 2460 tcttagagga tattgtttta acattgacct tatttgaaga tagggagatg attgaggaaa 2520 gacttaaaac atatgctcac ctctttgatg ataaggtgat gaaacagctt aaacgtcgcc 2580 gttatactgg ttggggacgt ttgtctcgaa aattgattaa tggtattagg gataagcaat 2640 ctggcaaaac aatattagat tttttgaaat cagatggttt tgccaatcgc aattttatgc 2700 agctgatcca tgatgatagt ttgacattta aagaagacat tcaaaaagca caagtgtctg 2760 gacaaggcga tagtttacat gaacatattg caaatttagc tggtagccct gctattaaaa 2820 aaggtatttt acagactgta aaagttgttg atgaattggt caaagtaatg gggcggcata 2880 agccagaaaa tatcgttatt gaaatggcac gtgaaaatca gacaactcaa aagggccaga 2940 aaaattcgcg agagcgtatg aaacgaatcg aagaaggtat caaagaatta ggaagtcaga 3000 ttcttaaaga gcatcctgtt gaaaatactc aattgcaaaa tgaaaagctc tatctctatt 3060 atctccaaaa tggaagagac atgtatgtgg accaagaatt agatattaat cgtttaagtg 3120 attatgatgt cgatcacatt gttccacaaa gtttccttaa agacgattca atagacaata 3180

129

SUBSTITUTE SHEET RULE 26 aggtcttaac gcgttctgat aaaaatcgtg gtaaatcgga taacgttcca agtgaagaag 3240 tagtcaaaaa gatgaaaaac tattggagac aacttctaaa cgccaagtta atcactcaac 3300 gtaagtttga taatttaacg aaagctgaac gtggaggttt gagtgaactt gataaagctg 3360 gttttatcaa acgccaattg gttgaaactc gccaaatcac taagcatgtg gcacaaattt 3420 tggatagtcg catgaatact aaatacgatg aaaatgataa acttattcga gaggttaaag 3480 tgattacctt aaaatctaaa ttagtttctg acttccgaaa agatttccaa ttctataaag 3540 tacgtgagat taacaattac catcatgccc atgatgcgta tctaaatgcc gtcgttggaa 3600 ctgctttgat taagaaatat ccaaaacttg aatcggagtt tgtctatggt gattataaag 3660 tttatgatgt tcgtaaaatg attgctaagt ctgagcaaga aataggcaaa gcaaccgcaa 3720 aatatttctt ttactctaat atcatgaact tcttcaaaac agaaattaca cttgcaaatg 3780 gagagattcg caaacgccct ctaatcgaaa ctaatgggga aactggagaa attgtctggg 3840 ataaagggcg agattttgcc acagtgcgca aagtattgtc catgccccaa gtcaatattg 3900 tcaagaaaac agaagtacag acaggcggat tctccaagga gtcaatttta ccaaaaagaa 3960 attcggacaa gcttattgct cgtaaaaaag actgggatcc aaaaaaatat ggtggttttg 4020 atagtccaac ggtagcttat tcagtcctag tggttgctaa ggtggaaaaa gggaaatcga 4080 agaagttaaa atccgttaaa gagttactag ggatcacaat tatggaaaga agttcctttg 4140 aaaaaaatcc gattgacttt ttagaagcta aaggatataa ggaagttaaa aaagacttaa 4200 tcattaaact acctaaatat agtctttttg agttagaaaa cggtcgtaaa cggatgctgg 4260 ctagtgccgg agaattacaa aaaggaaatg agctggctct gccaagcaaa tatgtgaatt 4320 ttttatattt agctagtcat tatgaaaagt tgaagggtag tccagaagat aacgaacaaa 4380 aacaattgtt tgtggagcag cataagcatt atttagatga gattattgag caaatcagtg 4440 aattttctaa gcgtgttatt ttagcagatg ccaatttaga taaagttctt agtgcatata 4500 acaaacatag agacaaacca atacgtgaac aagcagaaaa tattattcat ttatttacgt 4560 tgacgaatct tggagctccc gctgctttta aatattttga tacaacaatt gatcgtaaac 4620 gatatacgtc tacaaaagaa gttttagatg ccactcttat ccatcaatcc atcactggtc 4680 tttatgaaac acgcattgat ttgagtcagc taggaggtga ctgatggcca cgtgaactat 4740 atgattttcc gcagtata 4758

<210> 141

130

SUBSTITUTE SHEET RULE 26 <211> 276

<212> DNA

<213> Streptococcus pyogenes

<400> 141

attgatttga gtcagctagg aggtgactga tggccacgtg aactatatga ttttccgcag 60 tatattttag atgaagatta tttcttaata actaaaaata tggtataata ctcttaataa 120 atgcagtaat acaggggctt ttcaagactg aagtctagct gagacaaata gtgcgattac 180 gaaatttttt agacaaaaat agtctacgag gttttagagc tatgctgttt tgaatggtcc 240 caaaactgag accagtctcg gacgtccaaa ggtctc 276

<210> 142

<211> 10

<212> DNA

<213> Streptococcus pyogenes

<400> 142

ggtctccatt 10

<210> 143

<211> 10

<212> DNA

<213> Streptococcus pyogenes

<400> 143

ggtcccaaaa 10

<210> 144

<211> 452

<212> DNA

<213> Artificial

<220>

<223> Fragment 3, amplified from genomic DNA of S. pyrogenes strain

SF370

<400> 144

gagaccagtc tcggacgtcc aaaggtctcg ttttagagct atgctgtttt gaatggtccc 60 aaaacaacat tgccgatgat aacttgagaa agagggttaa taccagcagt cggatacctt 120 cctattcttt ctgttaaagc gttttcatgt tataataggc aaaagaagag tagtgtgatg 180 gaacaaacat tttttatgat taagccatat ggggttaagc aaggggaggt agttggagag 240 gttttacggt ggattgaacg cctaagattt acgtttaagc gattcgagct aagacaagct 300 agttcgaaat acttggctaa gcacgacgag gccttggtga taaacctttt gatcctaaac 360

131

SUBSTITUTE SHEET RULE 26 ttaaagctta catgacaagt ggtcctgttt taattgggat aattcttggg gactaaggtg 420 gtatcgtcca ttccgacagc atcgccagtc ac 452

<210> 145

<211> 9578

<212> DNA

<213> Artificial

Sequence of the final construct pNBlOO

<400> 145

gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt 60 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt 120 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga 180 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga 240 aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt 300 ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc 360 ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat 420 ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt 480 gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg 540 acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg agtgtatact 600 ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa 660 aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc 720 actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc 780 ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa 840 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc 900 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc 960 tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc 1020 gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac 1080 tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt 1140 gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt 1200 agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg 1260

132

SUBSTITUTE SHEET RULE 26 tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt 1320 cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc 1380 aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga tttcagtgca 1440 atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctc 1500 atgtttgaca gcttatcatc gataagcttt aatgcggtag tttatcacag ttaaattgct 1560 aacgcagtca ggcaccgtgt atgaaatcta acaatgcgct catcgtcatc ctcggcaccg 1620 tcaccctgga tgctgtaggc ataggcttgg ttatgccggt actgccgggc ctcttgcggg 1680 atccagaagt ctttttcttg cactgtttcc ttttctttat gatagtttac gaaatcatcc 1740 tgtggagctt agtaggttta gcaagatggc agcgcctaaa tgtagaatga taaaaggatt 1800 aagagattaa tttccctaaa aatgataaaa caagcgtttt gaaagcgctt gtttttttgg 1860 tttgcagtca gagtagaata gaagtatcaa aaaaagcacc gactcggtgc cactttttca 1920 agttgataac ggactagcct tattttaact tgctatgctg ttttgaatgg ttccaacaag 1980 attattttat aacttttata acaaataatc aaggagaaat tcaaagaaat ttatcagcca 2040 taaaacaata cttaatacta tagaatgata acaaaataaa ctacttttta aaagaatttt 2100 gtgttataat ctatttatta ttaagtattg ggtaatattt tttgaagaga tattttgaaa 2160 aagaaaaatt aaagcatatt aaactaattt cggaggtcat taaaactatt attgaaatca 2220 tcaaactcat tatggattta atttaaactt tttattttag gaggcaaaaa tggataagaa 2280 atactcaata ggcttagata tcggcacaaa tagcgtcgga tgggcggtga tcactgatga 2340 atataaggtt ccgtctaaaa agttcaaggt tctgggaaat acagaccgcc acagtatcaa 2400 aaaaaatctt ataggggctc ttttatttga cagtggagag acagcggaag cgactcgtct 2460 caaacggaca gctcgtagaa ggtatacacg tcggaagaat cgtatttgtt atctacagga 2520 gattttttca aatgagatgg cgaaagtaga tgatagtttc tttcatcgac ttgaagagtc 2580 ttttttggtg gaagaagaca agaagcatga acgtcatcct atttttggaa atatagtaga 2640 tgaagttgct tatcatgaga aatatccaac tatctatcat ctgcgaaaaa aattggtaga 2700 ttctactgat aaagcggatt tgcgcttaat ctatttggcc ttagcgcata tgattaagtt 2760 tcgtggtcat tttttgattg agggagattt aaatcctgat aatagtgatg tggacaaact 2820 atttatccag ttggtacaaa cctacaatca attatttgaa gaaaacccta ttaacgcaag 2880 tggagtagat gctaaagcga ttctttctgc acgattgagt aaatcaagac gattagaaaa 2940

133

SUBSTITUTE SHEET RULE 26 tctcattgct cagctccccg gtgagaagaa aaatggctta tttgggaatc tcattgcttt 3000 gtcattgggt ttgaccccta attttaaatc aaattttgat ttggcagaag atgctaaatt 3060 acagctttca aaagatactt acgatgatga tttagataat ttattggcgc aaattggaga 3120 tcaatatgct gatttgtttt tggcagctaa gaatttatca gatgctattt tactttcaga 3180 tatcctaaga gtaaatactg aaataactaa ggctccccta tcagcttcaa tgattaaacg 3240 ctacgatgaa catcatcaag acttgactct tttaaaagct ttagttcgac aacaacttcc 3300 agaaaagtat aaagaaatct tttttgatca atcaaaaaac ggatatgcag gttatattga 3360 tgggggagct agccaagaag aattttataa atttatcaaa ccaattttag aaaaaatgga 3420 tggtactgag gaattattgg tgaaactaaa tcgtgaagat ttgctgcgca agcaacggac 3480 ctttgacaac ggctctattc cccatcaaat tcacttgggt gagctgcatg ctattttgag 3540 aagacaagaa gacttttatc catttttaaa agacaatcgt gagaagattg aaaaaatctt 3600 gacttttcga attccttatt atgttggtcc attggcgcgt ggcaatagtc gttttgcatg 3660 gatgactcgg aagtctgaag aaacaattac cccatggaat tttgaagaag ttgtcgataa 3720 aggtgcttca gctcaatcat ttattgaacg catgacaaac tttgataaaa atcttccaaa 3780 tgaaaaagta ctaccaaaac atagtttgct ttatgagtat tttacggttt ataacgaatt 3840 gacaaaggtc aaatatgtta ctgaaggaat gcgaaaacca gcatttcttt caggtgaaca 3900 gaagaaagcc attgttgatt tactcttcaa aacaaatcga aaagtaaccg ttaagcaatt 3960 aaaagaagat tatttcaaaa aaatagaatg ttttgatagt gttgaaattt caggagttga 4020 agatagattt aatgcttcat taggtaccta ccatgatttg ctaaaaatta ttaaagataa 4080 agattttttg gataatgaag aaaatgaaga tatcttagag gatattgttt taacattgac 4140 cttatttgaa gatagggaga tgattgagga aagacttaaa acatatgctc acctctttga 4200 tgataaggtg atgaaacagc ttaaacgtcg ccgttatact ggttggggac gtttgtctcg 4260 aaaattgatt aatggtatta gggataagca atctggcaaa acaatattag attttttgaa 4320 atcagatggt tttgccaatc gcaattttat gcagctgatc catgatgata gtttgacatt 4380 taaagaagac attcaaaaag cacaagtgtc tggacaaggc gatagtttac atgaacatat 4440 tgcaaattta gctggtagcc ctgctattaa aaaaggtatt ttacagactg taaaagttgt 4500 tgatgaattg gtcaaagtaa tggggcggca taagccagaa aatatcgtta ttgaaatggc 4560 acgtgaaaat cagacaactc aaaagggcca gaaaaattcg cgagagcgta tgaaacgaat 4620

134

SUBSTITUTE SHEET RULE 26 cgaagaaggt atcaaagaat taggaagtca gattcttaaa gagcatcctg ttgaaaatac 4680 tcaattgcaa aatgaaaagc tctatctcta ttatctccaa aatggaagag acatgtatgt 4740 ggaccaagaa ttagatatta atcgtttaag tgattatgat gtcgatcaca ttgttccaca 4800 aagtttcctt aaagacgatt caatagacaa taaggtctta acgcgttctg ataaaaatcg 4860 tggtaaatcg gataacgttc caagtgaaga agtagtcaaa aagatgaaaa actattggag 4920 acaacttcta aacgccaagt taatcactca acgtaagttt gataatttaa cgaaagctga 4980 acgtggaggt ttgagtgaac ttgataaagc tggttttatc aaacgccaat tggttgaaac 5040 tcgccaaatc actaagcatg tggcacaaat tttggatagt cgcatgaata ctaaatacga 5100 tgaaaatgat aaacttattc gagaggttaa agtgattacc ttaaaatcta aattagtttc 5160 tgacttccga aaagatttcc aattctataa agtacgtgag attaacaatt accatcatgc 5220 ccatgatgcg tatctaaatg ccgtcgttgg aactgctttg attaagaaat atccaaaact 5280 tgaatcggag tttgtctatg gtgattataa agtttatgat gttcgtaaaa tgattgctaa 5340 gtctgagcaa gaaataggca aagcaaccgc aaaatatttc ttttactcta atatcatgaa 5400 cttcttcaaa acagaaatta cacttgcaaa tggagagatt cgcaaacgcc ctctaatcga 5460 aactaatggg gaaactggag aaattgtctg ggataaaggg cgagattttg ccacagtgcg 5520 caaagtattg tccatgcccc aagtcaatat tgtcaagaaa acagaagtac agacaggcgg 5580 attctccaag gagtcaattt taccaaaaag aaattcggac aagcttattg ctcgtaaaaa 5640 agactgggat ccaaaaaaat atggtggttt tgatagtcca acggtagctt attcagtcct 5700 agtggttgct aaggtggaaa aagggaaatc gaagaagtta aaatccgtta aagagttact 5760 agggatcaca attatggaaa gaagttcctt tgaaaaaaat ccgattgact ttttagaagc 5820 taaaggatat aaggaagtta aaaaagactt aatcattaaa ctacctaaat atagtctttt 5880 tgagttagaa aacggtcgta aacggatgct ggctagtgcc ggagaattac aaaaaggaaa 5940 tgagctggct ctgccaagca aatatgtgaa ttttttatat ttagctagtc attatgaaaa 6000 gttgaagggt agtccagaag ataacgaaca aaaacaattg tttgtggagc agcataagca 6060 ttatttagat gagattattg agcaaatcag tgaattttct aagcgtgtta ttttagcaga 6120 tgccaattta gataaagttc ttagtgcata taacaaacat agagacaaac caatacgtga 6180 acaagcagaa aatattattc atttatttac gttgacgaat cttggagctc ccgctgcttt 6240 taaatatttt gatacaacaa ttgatcgtaa acgatatacg tctacaaaag aagttttaga 6300

135

SUBSTITUTE SHEET RULE 26 tgccactctt atccatcaat ccatcactgg tctttatgaa acacgcattg atttgagtca 6360 gctaggaggt gactgatggc cacgtgaact atatgatttt ccgcagtata ttttagatga 6420 agattatttc ttaataacta aaaatatggt ataatactct taataaatgc agtaatacag 6480 gggcttttca agactgaagt ctagctgaga caaatagtgc gattacgaaa ttttttagac 6540 aaaaatagtc tacgaggttt tagagctatg ctattttgaa tggtcccaaa actgagacca 6600 gtctcggacg tccaaaggtc tcgttttaga gctatgctgt tttgaatggt cccaaaacaa 6660 cattgccgat gataacttga gaaagagggt taataccagc agtcggatac cttcctattc 6720 tttctgttaa agcgttttca tgttataata ggcaaaagaa gagtagtgtg atggaacata 6780 cattttttat gattaagcca tatggggtta agcaagggga ggtagttgga gaggttttac 6840 ggtggattga acgcctaaga tttacgttta agcgattcga gctaagacaa gctagttcga 6900 aatacttggc taagcacgac gaggccttgg tgataaacct tttgatccta aacttaaagc 6960 ttacatgaca agtggtcctg ttttaattgg gataattctt ggggactaag gtggtatcgt 7020 ccattccgac agcatcgcca gtcactatgg cgtgctgcta gcgctatatg cgttgatgca 7080 atttctatgc gcacccgttc tcggagcact gtccgaccgc tttggccgcc gcccagtcct 7140 gctcgcttcg ctacttggag ccactatcga ctacgcgatc atggcgacca cacccgtcct 7200 gtggatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc 7260 tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat 7320 gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc 7380 catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact 7440 gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgaccgatgc ccttgagagc 7500 cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat 7560 gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct gggtcatttt 7620 cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg 7680 aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga 7740 gaagcaggcc attatcgccg gcatggcggc cgacgcgctg ggctacgtct tgctggcgtt 7800 cgcgacgcga ggctggatgg ccttccccat tatgattctt ctcgcttccg gcggcatcgg 7860 gatgcccgcg ttgcaggcca tgctgtccag gcaggtagat gacgaccatc agggacagct 7920 tcaaggatcg ctcgcggctc ttaccagcct aacttcgatc attggaccgc tgatcgtcac 7980

136

SUBSTITUTE SHEET RULE 26 ggcgatttat gccgcctcgg cgagcacatg gaacgggttg gcatggattg taggcgccgc 8040 cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac 8100 ctgaatggaa gccggcggca cctcgctaac ggattcacca ctccaagaat tggagccaat 8160 caattcttgc ggagaactgt gaatgcgcaa accaaccctt ggcagaacat atccatcgcg 8220 tccgccatct ccagcagccg cacgcggcgc atctcgggca gcgttgggtc ctggccacgg 8280 gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact 8340 ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg 8400 tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa 8460 acgcggaagt cccctacgtg ctgctgaagt tgcccgcaac agagagtgga accaaccggt 8520 gataccacga tactatgact gagagtcaac gccatgagcg gcctcatttc ttattctgag 8580 ttacaacagt ccgcaccgct gtccggtagc tccttccggt gggcgcgggg catgactatc 8640 gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg 8700 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgccctgc 8760 accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca 8820 tctgtattaa cgaagcgcta accgttttta tcaggctctg ggaggcagaa taaatgatca 8880 tatcgtcaat tattacctcc acggggagag cctgagcaaa ctggcctcag gcatttgaga 8940 agcacacggt cacactgctt ccggtagtca ataaaccggt aaaccagcaa tagacataag 9000 cggctattta acgaccctgc cctgaaccga cgaccgggtc gaatttgctt tcgaatttct 9060 gccattcatc cgcttattat cacttattca ggcgtagcac caggcgttta agggcaccaa 9120 taactgcctt aaaaaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca 9180 ttaagcattc tgccgacatg gaagccatca cagacggcat gatgaacctg aatcgccagc 9240 ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag 9300 aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct 9360 gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa 9420 cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc 9480 cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta 9540 tcccatatca ccagctcacc gtctttcatt gccatacg 9578

<210> 146

137

SUBSTITUTE SHEET RULE 26 <211> 386

<212> DNA

<213> Artificial

<220>

<223> Amplified sequence with primer NB018 and NB019 from pNBlOO as

template

<400> 146

ccaaaactga gacctgctgc ggacgtccaa aggtctcgtt ttagagctat gctgttttga 60 atggtcccaa aacttgccga tgataacttg agaaagaggg ttaataccag cagtcggata 120 ccttcctatt ctttctgtta aagcgttttc atgttataat aggcaaattt tagatgaaga 180 ttatttctta ataactaaaa atatggtata atactcttaa taaatgcagt aatacagggg 240 cttttcaaga ctgaagtcta gctgagacaa atagtgcgat tacgaaattt tttagacaaa 300 aatagtctac gaggttttag agctatgctg ttttgaatgg tcccaaaact gaagagcgtc 360 tcggacgcag cgctcttcgt tttaga 386

<210> 147

<211> 744

<212> DNA

<213> Artificial

<220>

<223> Modified CRISPR array in pNB200

<400> 147

ggtgactgat ggccacgtga actatatgat tttccgcagt atattttaga tgaagattat 60 ttcttaataa ctaaaaatat ggtataatac tcttaataaa tgcagtaata caggggcttt 120 tcaagactga agtctagctg agacaaatag tgcgattacg aaatttttta gacaaaaata 180 gtctacgagg ttttagagct atgctatttt gaatggtccc aaaactgaga cctgctgcgg 240 acgtccaaag gtctcgtttt agagctatgc tgttttgaat ggtcccaaaa cttgccgatg 300 ataacttgag aaagagggtt aataccagca gtcggatacc ttcctattct ttctgttaaa 360 gcgttttcat gttataatag gcaaatttta gatgaagatt atttcttaat aactaaaaat 420 atggtataat actcttaata aatgcagtaa tacaggggct tttcaagact gaagtctagc 480 tgagacaaat agtgcgatta cgaaattttt tagacaaaaa tagtctacga ggttttagag 540 ctatgctgtt ttgaatggtc ccaaaactga agagcgtctc ggacgcagcg ctcttcgttt 600 tagagctatg ctgttttgaa tggtcccaaa acaacattgc cgatgataac ttgagaaaga 660 gggttaatac cagcagtcgg ataccttcct attctttctg ttaaagcgtt ttcatgttat 720

138

SUBSTITUTE SHEET RULE 26 aataggcaaa agaagagtag tgtg

<210> 148

<211> 23

<212> DNA

<213> Artificial

<220>

<223> Primer sequence <400> 148

ccaactacct ccccttgctt

<210> 149

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 149

ggtgactgat ggccacgt

<210> 150

<211> 23

<212> DNA

<213> Artificial

<220>

<223> Primer sequence <400> 150

gggctggcaa gccacgtttg gtg

<210> 151

<211> 23

<212> DNA

<213> Artificial

<220>

<223> Primer sequence <400> 151

ccgggagctg catgtgtcag agg

<210> 152

<211> 43

<212> DNA

<213> Artificial <220>

<223> Primer sequence

<400> 152

attgaaaaag gaagagtatg gaattgccca atattatgca ccc 43

<210> 153

<211> 38

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 153

agtcccgcta ggtctcaacc gtcagcgcag cttgtcgg 38

<210> 154

<211> 42

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 154

attgaaaaag gaagagtatg cgtgtattag ccttatcggc tg 42

<210> 155

<211> 55

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 155

agtcccgcta ggtctcaacc gctagggaat aattttttcc tgtttgagca cttct 55

<210> 156

<211> 47

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 156

attgaaaaag gaagagtatg cgttattttc gcctgtgtat tatctcc 47 <210> 157

<211> 41

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 157

agtcccgcta ggtctcaacc gttagcgttg ccagtgctcg

<210> 158

<211> 57

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 158

attgaaaaag gaagagtatg ttaaaagtta ttagtagttt attggtctac atgaccg 57

<210> 159

<211> 48

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 159

agtcccgcta ggatgacctg gctgaccgct actcggcgac tgagcgat

<210> 160

<211> 43

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 160

attgaaaaag gaagagtatg tcactgtatc gccgtctagt tct 43

<210> 161

<211> 39

<212> DNA

<213> Artificial

<220>

<223> KPC-3 reverse primer <400> 161

agtcccgcta ggtctcaacc gttactgccc gttgacgcc 39

<210> 162

<211> 60

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 162

attgaaaaag gaagagtatg agcaagttat ctgtattctt tatatttttg ttttgtagca

<210> 163

<211> 61

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 163

agtcccgcta ggtctcaacc gttagttgct tagttttgat ggttttttac tttcgtttaa

<210> 164

<211> 44

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 164

attgaaaaag gaagagtatg gttaaaaaat cactgcgcca 44

<210> 165

<211> 47

<212> DNA

<213> Artificial

<220>

<223> Primer sequence

<400> 165

agtcccgcta ggtctcaacc gttacaaacc gtcggtgacg attttag 47

<210> 166

<211> 834 <212> DNA

<213> Unknown

<220>

<223> NDM-1 sequence

<400> 166

accgtcagcg cagcttgtcg gccatgcggg ccgtatgagt gattgcggcg cggctatcgg 60 gggcggaatg gctcatcacg atcatgctgg ccttggggaa cgccgcacca aacgcgcgcg 120 ctgacgcggc gtagtgctca gtgtcggcat caccgagatt gccgagcgac ttggccttgc 180 tgtccttgat caggcagcca ccaaaagcga tgtcggtgcc gtcgatccca acggtgatat 240 tgtcactggt gtggccgggg ccggggtaaa ataccttgag cgggccaaag ttgggcgcgg 300 ttgctggttc gacccagcca ttggcggcga aagtcaggct gtgttgcgcc gcaaccatcc 360 cctcttgcgg ggcaagctgg ttcgacaacg cattggcata agtcgcaatc cccgccgcat 420 gcagcgcgtc cataccgccc atcttgtcct gatgcgcgtg agtcaccacc gccagcgcga 480 ccggcaggtt gatctcctgc ttgatccagt tgaggatctg ggcggtctgg tcatcggtcc 540 aggcggtatc gaccaccagc acgcggccgc catccctgac gatcaaaccg ttggaagcga 600 ctgccccgaa acccggcatg tcgagatagg aagtgtgctg ccagacattc ggtgcgagct 660 ggcggaaaac cagatcgcca aaccgttggt cgccagtttc catttgctgg ccaatcgtcg 720 ggcggatttc accgggcatg cacccgctca gcatcaatgc agcggctaat gcggtgctca 780 gcttcgcgac cgggtgcata atattgggca attccatact cttccttttt caat 834

<210> 167

<211> 819

<212> DNA

<213> Unknown

OXA-48 sequence

<400> 167

accgctaggg aataattttt tcctgtttga gcacttcttt tgtgatggct tggcgcagcc 60 ctaaaccatc cgatgtgggc atatccatat tcatcgcaaa aaaccacaca ttatcatcaa 120 gttcaaccca accgacccac cagccaatct taggttcgat tctagtcgag tatccagttt 180 tagcccgaat aatatagtca ccattggctt cggtcagcat ggcttgtttg acaatacgct 240 ggctgcgctc cgatacgtgt aacttattgt gatacagctt tcttaaaaag ctgatttgct 300 ccgtggccga aattcgaata ccaccgtcga gccagaaact gtctacattg cccgaaatgt 360

143

SUBSTITUTE SHEET RULE 26 cctcattacc ataatcgaaa gcatgtagca tcttgctcat acgtgcctcg ccaatttggc 420 gggcaaattc ttgataaaca ggcacaactg aatatttcat cgcggtgatt agattatgat 480 cgcgattcca agtggcgata tcgcgcgtct gtccatccca cttaaagact tggtgttcat 540 ccttaaccac gcccaaatcg agggcgatca agctattggg aattttaaag gtagatgcgg 600 gtaaaaatgc ttggttcgcc cgtttaagat tattggtaaa tccttgctgc ttattctcat 660 tccagagcac aactacgccc tgtgatttat gttcagtaaa gtgagcattc caacttttgt 720 tttcttgcca ttcctttgct accgcaggca ttccgataat cgatgccacc aaaaacacag 780 ccgataaggc taatacacgc atactcttcc tttttcaat 819

<210> 168

<211> 882

<212> DNA

<213> Unknown

<220>

<223> SHV-1 sequence

<400> 168

accgttagcg ttgccagtgc tcgatcagcg ccgcgccgat cccggcgatt tgctgatttc 60 gctcggccat gctcgccggc gtatcccgca gataaatcac cacaatccgc tctgctttgt 120 tattcgggcc aagcagggcg acaatcccgc gcgcaccccg tttggcagct ccggtcttat 180 cggcgataaa ccagcccgcc ggcagcacgg agcggatcaa cggtccggcg acccgatcgt 240 ccaccatcca ctgcagcagc tgccgttgcg aacgggcgct cagacgctgg ctggtcagca 300 gcttgcgcag ggtcgcggcc atgctggccg gggtagtggt gtcgcgggcg tcgccgggaa 360 gcgcctcatt cagttccgtt tcccagcggt caaggcgggt gacgttgtcg ccgatctggc 420 gcaaaaaggc agtcaatcct gcggggccgc cgacggtggc cagcagcaga ttggcggcgc 480 tgttatcgct catggtaatg gcggcggcac agagttcgcc gaccgtcatg ccgtcggcaa 540 ggtgtttttc gctgaccggc gagtagtcca ccagatcctg ctggcgatag tggatctttc 600 gctccagctg ttcgtcaccg gcatccaccc gcgccagcac tgcgccgcag agcactactt 660 taaaggtgct catcatggga aagcgttcat cggcgcgcca ggcggtcagc gtgcggccgc 720 tggccagatc catttctatc atgcctacgc tgcccgacag ctggctttcg cttagtttaa 780 tttgctcaag cggctgcggg ctggcgtgta ccgccagcgg cagggtggct aacagggaga 840 taatacacag gcgaaaataa cgcatactct tcctttttca at 882

144

SUBSTITUTE SHEET RULE 26 <210> 169

<211> 822

<212> DNA

<213> Unknown

<220>

<223> VIM-1 sequence

<400> 169

accgctactc ggcgactgag cgatttttgt gtgctttgac aacgttcgct gtgtgctgga 60 gcaagtctag accgcccggt agaccgtgcc cgggaatgac gacctctgct tccgggtagt 120 gtttttgaat ccgctcaacg gaggtgggcc attcagccag atcggcatcg gccacgttcc 180 ccgcagacgt gcttgacaac tcatgaacgg cacaaccacc gtatagcacg ttcgctgacg 240 ggacgtatac aaccagattg tcggtcgaat gcgcagcacc aggatagaag agctctactg 300 gaccgaagcg cactgcgtcc ccgctcgatg agagtccttc tagagaatgc gtgggaatct 360 cgttcccctc tgcctcggct agccggcgtg tcgacggtga tgcgtacgtt gccaccccag 420 ccgcccgaag gacatcaacg ccgccgacgc ggtcgtcatg aaagtgcgtg gagactgcac 480 gcgttacggg aagtccaatt tgcttttcaa tctccgcgag aagtgccgct gtgtttttcg 540 caccccacgc tgtatcaatc aaaagcaact catcaccatc acggacaatg agaccattgg 600 acgggtagac cgcgccatca aacgactgcg ttgcgatatg cgaccaaaca ccatcggcaa 660 tctggtaaag tcggacctct ccgaccggaa tttcgttgac tgtcggatac tcaccactcg 720 gctccccgga atgggctaac ggacttgcga cagccatgac agacgcggtc atgtagacca 780 ataaactact aataactttt aacatactct tcctttttca at 822

<210> 170

<211> 903

<212> DNA

<213> Unknown

<220>

<223> KPC-3 sequence

<400> 170

accgttactg cccgttgacg cccaatccct cgagcgcgag tctagccgca gcggcgatga 60 cggcctcgct gtacttgtca tccttgttag gcgcccgggt gtagacggcc aacacaatag 120 gtgcgcgccc agtgggccag acgacggcat agtcatttgc cgtgccatac actccgcagg 180 ttccggtttt gtctccgact gcccagtctg ccggcaccgc cgcgcggatg cggtggttgc 240

145

SUBSTITUTE SHEET RULE 26 cggtcgtgtt tccctttagc caatcaacaa actgctgccg ctgcggcgca gccagtgcag 300 agcccagtgt cagtttttgt aagctttccg tcacggcgcg cggcgatgag gtatcgcgcg 360 catcgcctgg gatggcggag ttcagctcca gctcccagcg gtccagacgg aacgtggtat 420 cgccgataga gcgcatgaag gccgtcagcc cggccgggcc gcccaactcc ttcagcaaca 480 aattggcggc ggcgttatca ctgtattgca cggcggccgc ggacagctcc gccaccgtca 540 tgcctgttgt cagatatttt tccgagatgg gtgaccacgg aaccagcgca tttttgccgt 600 aacggatggg tgtgtccagc aagccggcct gctgctggct gcgagccagc acagcggcag 660 caagaaagcc cttgaatgag ctgcacagtg ggaagcgctc ctcagcgcgg taacttacag 720 ttgcgcctga gccggtatcc atcgcgtaca caccgatgga gccgccaaag tcctgttcga 780 gtttagcgaa tggttccgcg acgaggttgg tcagcgcggt ggcagaaaag ccagccagcg 840 gccatgagag acaagacagc agaactagac ggcgatacag tgacatactc ttcctttttc 900 aat 903

<210> 171

<211> 762

<212> DNA

<213> Unknown

<220>

<223> IMP-4 sequence

<400> 171

accgttagtt gcttagtttt gatggttttt tactttcgtt taacccttta accgcctgct 60 ctaatgtaag tttcaagagt gatgcgtctc cagcttcact gtgacttgga acaaccagtt 120 ttgccttacc atatttggat attaataatt tagcggactt tggccaagct tctaaatttg 180 cgtcacccaa attacctaga ccgtacggtt taataaaaca accaccgaat aatattttcc 240 tttcaggcag ccaaactact aggttatctg gagtgtgtcc tgggcctgga taaaaaactt 300 caattttatt tttaactagc caatagttaa ccccgccaaa tgaattttta gcttgaacct 360 taccgtcttt tttaagcagc tcattagtta attcagacgc atacgtgggg atggattgag 420 aattaagcca ctctattccg cccgtgctgt cactatgaaa atgagaggaa atactgcctt 480 ttattttata gccacgttcc acaaaccaag tgactaactt ttcagtatct ttagccgtaa 540 atggagtgtc aattagataa gcttcagcat ctacaagaac aaccaaacca tgtttaggaa 600 caacgcccca cccgttaact tcttcaaacg aagtatgaac ataaacgcct tcatcaagtt 660

146

SUBSTITUTE SHEET RULE 26 tttcaatttt taaatctggc aaagactctg ctgcggtagc aatgctacaa aacaaaaata taaagaatac agataacttg ctcatactct tcctttttca at

<210> 172

<211> 897

<212> DNA

<213> Unknown

CTX-M-15 sequence

<400> 172

accgttacaa accgtcggtg acgattttag ccgccgacgc taatacatcg cgacggcttt 60 ctgccttagg ttgaggctgg gtgaagtaag tgaccagaat cagcggcgca cgatcttttg 120 gccagatcac cgcgatatcg ttggtggtgc catagccacc gctgccggtt ttatccccca 180 caacccagga agcaggcagt ccagcctgaa tgctcgctgc accggtggta ttgcctttca 240 tccatgtcac cagctgcgcc cgttggctgt cgcccaatgc tttacccagc gtcagattcc 300 gcagagtttg cgccattgcc cgaggtgaag tggtatcacg cggatcgccc ggaatggcgg 360 tgtttaacgt cggctcggta cggtcgagac ggaacgtttc gtctcccagc tgtcgggcga 420 acgcggtgac gctagccggg ccgccaacgt gagcaatcag cttattcatc gccacgttat 480 cgctgtactg tagcgcggcc gcgctaagct cagccagtga catcgtccca ttgacgtgct 540 tttccgcaat cggattatag ttaacaaggt cagatttttt gatctcaact cgctgattta 600 acagattcgg ttcgctttca cttttcttca gcaccgcggc cgcggccatc actttactgg 660 tgctgcacat cgcaaagcgc tcatcagcac gataaagtat ttgcgaatta tctgctgtgt 720 taatcaatgc cacacccagt ctgcctcccg actgccgctc taattcggca agtttttgct 780 gtacgtccgc cgtttgcgca tacagcggca cacttcctaa caacagcgtg acggttgccg 840 tcgccatcag cgtgaactgg cgcagtgatt ttttaaccat actcttcctt tttcaat 897

147

SUBSTITUTE SHEET RULE 26