Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
METHOD FOR THE GENERATION OF A NON-CRISPR BACTERIOPHAGE INSENSITIVE MUTANT USING AN ANTI-CRISPR PROTEIN
Document Type and Number:
WIPO Patent Application WO/2020/101486
Kind Code:
A1
Abstract:
The invention provides a method for the generation of a bacteriophage insensitive mutant from parent strain cells comprising an active CRISPR-Cas system, the method comprising the stages: -an anti-CRISPR protein exposure stage comprising exposing the parent strain cells to an anti-CRISPR protein suitable for inhibiting the CRISPR-Cas system, wherein the anti-CRISPR protein comprises a protein comprising an amino acid sequence, wherein the amino acid sequence has at least 30% sequence identity with the amino acid sequence of SEQ ID NO:1with respect to a sequence alignment between the amino acid sequence and SEQ ID NO:1, and wherein the sequence alignment has a length ≥ 50% of the sequence length of SEQ ID NO:1; -a bacteriophage exposure stage comprising exposing the parent strain cells to a bacteriophage; -a selection stage comprising selecting the CRISPR-independent bacteriophage insensitive mutant from cells having survived the bacteriophage exposure stage.

Inventors:
DE NÓBREGA FRANKLIN LUZIA (NL)
BROUNS STAN JOHAN JOZEF (NL)
Application Number:
PCT/NL2019/050738
Publication Date:
May 22, 2020
Filing Date:
November 12, 2019
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
UNIV DELFT TECH (NL)
International Classes:
C12N15/01; A23C9/123; A23C19/032; C07K14/005; C07K14/315; C12N1/20; C12R1/46
Domestic Patent References:
WO2018197495A12018-11-01
WO2016012552A12016-01-28
WO2015124718A12015-08-27
WO2018142416A12018-08-09
WO2015124718A12015-08-27
WO2018197495A12018-11-01
Other References:
MCDONNELL B. ET AL: "Generation of Bacteriophage-Insensitive Mutants of Streptococcus thermophilus via an Antisense RNA CRISPR-Cas Silencing Appraoch", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 84:e1733-17, no. 4, 27 November 2017 (2017-11-27), pages 1 - 14, XP055596146
HYNES A. P. ET AL: "An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9", NATURE MICROBIOLOGY, vol. 2, no. 10, 7 August 2017 (2017-08-07), pages 1374 - 1380, XP036429527
HYNES A. P. ET AL: "Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins", NATURE COMMUNICATIONS, vol. 9:2919, 25 July 2018 (2018-07-25), pages 1 - 10, XP055595982
PAWLUK A. ET AL: "Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species", NATURE MICROBIOLOGY, vol. 1:16085, 13 June 2016 (2016-06-13), pages 1 - 6, XP055474868
QUIBERONI A. ET AL: "Streptococcus thermophilus bacteriophages", INTERNATIONAL DAIRY JOURNAL, vol. 20, no. 10, 1 October 2010 (2010-10-01), pages 657 - 664, XP027174041
MILLS S. ET AL: "Efficient method for generation of bacteriophage insensitive mutants of Streptococcus thermophilus yoghurt and mozzarella strains", JOURNAL OF MICROBIOLOGICAL METHODS, vol. 70, no. 1, 24 April 2007 (2007-04-24), pages 159 - 164, XP022130113
MILLS ET AL., JOURNAL OF MICROBIOLOGICAL METHODS, vol. 70, 2007, pages 159 - 164
"NCBI", Database accession no. NP -049960.1
"GenBank", Database accession no. ARU 13887.1
SOMKUTISTEINBERG, GENETIC TRANSFORMATION OF STREPTOCOCCUS THERMOPHILUS, 1988
RAN, GENOME ENGINEERING USING THE CRISPR-CAS9 SYSTEM, 2013
MILLS ET AL., EFFICIENT METHOD FOR GENERATION OF BACTERIOPHAGE INSENSITIVE MUTANTS OF STREPTOCOCCUS THERMOPHILUS YOGHURT AND MOZZARELLA STRAINS, 2007
SWARTS ET AL., CRISPR INTERFERENCE DIRECTS STRAND SPECIFIC SPACER ACQUISITION, 2012
SURESH ET AL.: "Cell-Penetrating Peptide-Mediated Delivery of Cas9 Protein and Guide RNA for Genome Editing", METHODS IN MOLECULAR BIOLOGY, 2017
KATOHSTANDLEY: "Improvements in Performance and Usability", MAFFT MULTIPLE SEQUENCE ALIGNMENT SOFTWARE, 2013
Attorney, Agent or Firm:
EDP PATENT ATTORNEYS B.V. (NL)
Download PDF:
Claims:
CLAIMS:

1. A method for the generation of a bacteriophage insensitive mutant from parent strain cells comprising an active CRISPR-Cas system, the method comprising the stages:

- an anti-CRISPR protein exposure stage comprising exposing the parent strain cells to an anti-CRISPR protein suitable for inhibiting the CRISPR-Cas system, wherein the anti- CRISPR protein comprises a protein comprising an amino acid sequence, wherein the amino acid sequence has at least 30% sequence identity with the amino acid sequence of SEQ ID NO: 1 with respect to a sequence alignment between the amino acid sequence and SEQ ID NO: l, and wherein the sequence alignment has a length > 50% of the sequence length of SEQ ID NO: 1;

- a bacteriophage exposure stage comprising exposing the parent strain cells to a bacteriophage;

- a selection stage comprising selecting the bacteriophage insensitive mutant from cells having survived the bacteriophage exposure stage.

2. The method according to claim 1, wherein the parent strain cells comprise a bacterium belonging to the group consisting of the genera Lactococcus, Leuconostoc, Lactobacillus, Escherichia and Streptococcus.

3. The method according to any one of the preceding claims, wherein the bacteriophage comprises one or more bacteriophages selected from the order Caudovirales.

4. The method according to any one of the preceding claims, wherein the anti- CRISPR protein exposure stage comprises exposing the parent strain cells to the anti- CRISPR protein in a concentration of at least 10 mM of the anti-CRISPR protein.

5. The method according to any one of the preceding claims, wherein the anti- CRISPR protein exposure stage further comprises exposing the parent strain cells to an auxiliary inhibitor, wherein the auxiliary inhibitor comprises one or more of an antisense RNA and an anti-CRISPR-associated protein.

6. The method according to any one of the preceding claims, wherein the anti- CRISPR protein exposure stage comprises exposing the parent strain cells to a cell- penetrating peptide.

7. The method according to any one of the preceding claims, wherein the anti- CRISPR protein exposure stage comprises providing the anti-CRISPR protein to the parent strain cells via direct protein addition.

8. The method according to any one of the preceding claims, wherein the parent strain cells comprise a plurality of (active) CRISPR-Cas systems, and wherein the anti- CRISPR protein inhibits all of the plurality of CRISPR-Cas systems.

9. The method according to any one of the preceding claims, wherein the selection stage further comprises comparing CRISPR arrays of the surviving cells with CRISPR arrays of the parent strain cells and selecting the bacteriophage insensitive mutant based on having an identical CRISPR array to the CRISPR array of the parent strain cells.

10. The method according to any one of the preceding claims, wherein the parent strain cells comprise a plasmid, wherein the plasmid comprises a gene, wherein the gene encodes the anti-CRISPR protein.

11. The method according to claim 10, wherein the method further comprises curing the surviving cells of the plasmid.

12. The method according to any one of the preceding claims, wherein the bacteriophage exposure stage comprises exposing the parent strain cells to the bacteriophage in a concentration >10J pfu/ml of the bacteriophage.

13. A bacteriophage insensitive mutant obtained using the method according to any one of the preceding claims.

14. The bacteriophage insensitive mutant according to claim 13, wherein the bacteriophage insensitive mutant has a reduced sensitivity to one or more bacteriophages selected from the order Caudovirales. 15. Use of the bacteriophage insensitive mutant according to any one of the preceding claims 13-14 in a process for producing a dairy product. 16. A food product comprising the bacteriophage insensitive mutant according to any one of the preceding claims 13-14.

17. A dairy product comprising the bacteriophage insensitive mutant according to any one of the preceding claims 13-14.

18. A probiotic product comprising the bacteriophage insensitive mutant according to any one of the preceding claims 13-14.

Description:
Method for the generation of a non-CRISPR-Cas-mediated bacteriophage insensitive mutant using an anti-CRISPR protein

FIELD OF THE INVENTION

The invention relates to a method for generating a non-CRISPR-Cas- mediated bacteriophage insensitive mutant. The invention further relates to the bacteriophage insensitive mutant. The invention further relates to a use of the bacteriophage insensitive mutant.

BACKGROUND OF THE INVENTION

Methods providing non-CRISPR-mediated bacteriophage insensitive mutants are known in the art. For example, WO2015124718A1 describes that bacteriophage insensitive mutants (hereinafter also abbreviated as “BIMs”) of three Streptococcus thermophihis parent strains were generated and characterized for phage sensitivity, sedimentation rate, cell chain length, phage adsorption and CRISPR loci alterations. Several BIMs showed an altered sedimentation phenotype as well as an increase cell chain length, reduced phage sensitivity, reduced phage adsorption and 100% identity in three CRISPR loci.

WO2018197495 describes a method to favor the screening of a bacterial mechanism providing resistance against a virulent phage other than a given class 2 type II CRISPR-Cas mediated resistance, comprising: providing a bacterial strain, the genome of which contains a given class 2 type II CRISPR-Cas system, which is known to be active against target nucleic acids; expressing in said bacterial strain a gene encoding a protein which interferes with a function of said given class 2 type II CRISPR-Cas system; exposing said bacterial strain of step 2) to a viailent phage; and selecting bacteriophage-insensitive mutants (BIMs).

SUMMARY OF THE INVENTION

A large range of products including, for example, food, feed, pharmaceuticals, commodity chemicals, detergents, biofuels, and textiles may be produced industrially by large-scale fermentation of organic substrates using micro-organisms, especially bacteria. These large-scale fermentations may, however, regularly become contaminated with a bacteriophage causing fermentation failure, which may result in production delays and/or economic setbacks.

For example, bacteriophages targeting lactic acid bacteria, such as Streptococcus thermophilus , may be a persistent and costly problem in the dairy industry. Several approaches may have been used to reduce the impact of bacteriophage infection, among which the generation of BIMs to use as starter strains. Ideally, BIMs should be: resistant to genetically distinct bacteriophages, i.e., survive exposure to genetically distinct bacteriophages; have technological properties identical to the parental strain, for example, a Streptococcus BIM may ideally be substantially identical with regards to one or more of exopolysaccharide (EPS) production, growth kinetics in milk medium, urease activity and galactose fermentation; have easy genetic and phenotypic characterization; and have robustness. The latter may refer to the ability of a BIM to survive exposure to bacteriophages at high concentrations, repeated bacteriophage challenges, and challenges with genetically distinct bacteriophages.

The prior art describes several methods for the generation of BIMs. A prior art method may comprise exposing a parent strain to a bacteriophage, incubating the surviving cells in a growth medium, exposing the grown cells to the bacteriophage in a plurality of passages in a second growth medium, plating the cells after passaging to select and further assess individual (BIM) colonies. This process may then be repeated to obtain resistance against a second bacteriophage.

It has been shown that robustness of the BIM may be dependent on the mechanisms conferring bacteriophage resistance. For example, the most characterized bacteriophage resistance mechanism of S. thermophilus may be CRISPR-Cas (Type II-A CRISPR1 and CRISPR3); but this system has been shown unpredictable and/or instable with regards to bacteriophage resistance, and bacteriophages have been shown to (rapidly) overcome this CRISPR-Cas-mediated resistance. For example, bacteriophages may acquire one or more mutations in a recognition sequence (protospacer) or protospacer adjacent motif (PAM), forcing the host to acquire a new spacer in order to regain CRISPR-Cas-mediated insensitivity.

Hence, non-CRISPR-Cas-mediated BIMs (hereinafter also abbreviated as “nCBIMs”) of A thermophilus , as well as of other organisms, may be preferred, for example as non-CRISPR-mediated BIMs may be more robust towards mutations in bacteriophages as the resistance mechanism may not directly depend on the recognition of a protospacer as for CRISPR-Cas-mediated insensitivity. Non-CRISPR-Cas-mediated insensitivity may further provide a broader resistance, i.e., CRISPR-Cas-mediated insensitivity may protect against a specific bacteriophage, whereas the non-CRISPR-Cas-mediated insensitivity may protect against a range of bacteriophages.

The prior art further includes a strategy for the selection of nCBIMs which may involve using an antisense RNA CRISPR-Cas silencing approach. This strategy may involve silencing one or more CRISPR-associated genes (such as Cas9 and Csn2) in the CRISPRl and CRISPR3 systems of S. thermophilus using antisense RNA. However, CRISPR spacer acquisitions still occurred (although they may have been fewer), and CRISPR-Cas-mediated BIMs (hereinafter also abbreviated as“CBIMs”) may still have been obtained.

The prior art methods may generate CBIMs as well as nCBIMs, especially substantially more CBIMs with respect to nCBIMs. Further, prior art methods involving mutagenesis may generate large numbers of DNA mutations in genes unrelated to bacteriophage insensitivity. Hence, the prior art methods may require substantial screening efforts to select suitable nCBIMs. Further, the prior art methods may involve the formation of double-stranded RNA (dsRNA), which may have undesired effects on the cells. Yet further, the BIMs generated by prior art methods may be genetically modified organisms, which may limit their (industrial) applicability due to regulatory requirements and/or consumer acceptance. Yet further, the prior art methods may require substantial adaptation for different parent strains; for example, the antisense RNA may have to be designed for a specific strain. Yet further, the prior art methods may not successfully deactivate the CRISPR-Cas systems of the targeted organisms due to (synonymous) mutations in CRISPR- associated genes.

Hence, it is an aspect of the invention to provide an alternative method for generating bacteriophage insensitive mutants, which preferably further at least partly obviates one or more of above-described drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

Therefore, in a first aspect, the invention provides a method for the generation of a non-CRISPR-Cas-mediated bacteriophage insensitive mutant (nCBIM) from parent strain cells comprising an active CRISPR-Cas system, the method comprising the stages: - an anti-CRISPR protein exposure stage comprising exposing the parent strain cells to an anti-CRISPR protein suitable for inhibiting the CRISPR-Cas system; and - a bacteriophage exposure stage comprising exposing the parent strain cells to a bacteriophage, especially a bacteriophage capable of infecting (at least some of) the parent strain cells. In embodiments, the method may further comprise a selection stage comprising selecting the bacteriophage insensitive mutant, especially the CRISPR-independent bacteriophage insensitive mutant, from cells having survived the bacteriophage exposure stage. Especially, the parent strain cells may comprise Streptococcus thermophilus (cells).

The invention as described herein may facilitate generating an nCBIM with a reduced, or even removed, need for screening against CBIMs. The anti-CRISPR-protein (ACR) may substantially inhibit, such as completely inhibit, the CRISPR-Cas system of the parent strain cells, suggesting that the cells surviving the bacteriophage exposure stage did not do so due to a CRISPR-Cas-mediated insensitivity. The surviving cells may thus be likely to have a non-CRISPR-C as-mediated insensitivity.

In specific embodiments, the invention provides a method for the generation of a bacteriophage insensitive mutant from parent strain cells comprising an active CRISPR- Cas system, the method comprising the stages: an anti-CRISPR protein exposure stage comprising exposing the parent strain cells to an anti-CRISPR protein suitable for inhibiting the CRISPR-Cas system; a bacteriophage exposure stage comprising exposing the parent strain cells to a bacteriophage; a selection stage comprising selecting the bacteriophage insensitive mutant from cells having survived the bacteriophage exposure stage.

The use of an anti-CRISPR protein may facilitate the generation of a BIM of a parent strain, especially of a S. thermophilus strain, wherein the BIM may be resistant to phage infection through a non-CRISPR-Cas-mediated mechanism. To do so, the parent strain, especially the S. thermophilus strain, may be transformed with a plasmid expressing the ACR, and then be subjected to a protocol for BIM generation, such as a standard protocol known to the person skilled in the art, such as using the protocol described by Mills et al. 2007, Journal of Microbiological Methods, 70 159-164, which is herein incorporated by reference. Especially, an ACR may target both the CRISPR1 and CRISPR3 (systems) of A thermophilus , and robust nCBIMs may be obtained exhibiting stable phage resistance.

Further, the use of an ACR may provide an increased likelihood of successful CRISPR-Cas system inhibition relative to an antisense RNA approach, i.e., inhibition of the CRISPR-Cas system with an anti-CRISPR protein may be more robust to mutations in the CRISPR-Cas system than an antisense RNA approach. For example, due to the redundancy in the codon table, a parent strain cell comprising one or more (synonymous) mutations in a targeted CRISPR-Cas gene may be capable of avoiding successful inhibition of its CRISPR-Cas system by an antisense RNA but may not be capable of avoiding successful inhibition of its CRISPR-Cas system by an ACR. The term“synonymous mutation” refers to a substitution of a nucleobase in a gene by another nucleobase, wherein the substitution does not affect the corresponding amino acid sequence. For example, if the third adenine in codon AAA is substituted by a guanine (AAG), the encoded amino acid (lysine) is not changed. The increased likelihood of inhibition of the CRISPR-Cas system may result in an increased likelihood of acquiring nCBIMs with respect to CBIMs.

Further, the use an ACR may reduce, especially prevent, plasmid loss via the CRISPR-Cas system. For example, the parent strain may comprise a (naturally transduced) plasmid, especially a beneficial plasmid, containing one or more genes encoding phage receptors. Hence, the parent strain, when exposed to bacteriophages taking advantage of the plasmid-encoded phage receptors could lose the (otherwise beneficial) plasmid for improved bacteriophage resistance. Such (undesired) plasmid loss may involve the CRISPR-Cas system and may thus be reduced, especially prevented, via the method according to the invention.

The invention may further facilitate the generation of non-GMO nCBIMs (see further below).

A bacteriophage (also:“phage”) is a viais that infects and replicates within prokaryotes, which may (typically) result in the death of a successfully infected prokaryotic cell.

The CRISPR-Cas system (also “CRISPR system”) (clustered regularly interspaced short palindromic repeats-CRISPR associated proteins) is an adaptive prokaryotic immune system. The CRISPR-Cas system comprises a CRISPR array and CRISPR-associated proteins (Cas proteins). The CRISPR array (also:“CRISPR cassette” and“CRISPR repeat”) comprises a plurality of repeated sequences (termed“repeats”) interspersed and followed by spacers. A spacer is a short piece of DNA homologous to a target sequence, such as a bacteriophage sequence, a plasmid sequence, and/or a genome sequence. The CRISPR-Cas system, especially the Cas proteins, may be configured to recognize target sequences corresponding to the spacers in the CRISPR array and to cleave polynucleotides comprising any one of the target sequences, thereby potentially stopping a bacteriophage infection. In addition, the CRISPR-Cas system, especially the Cas proteins, may be configured to integrate a new spacer into the CRISPR array corresponding to an encountered bacteriophage (or plasmid). The integration of a new spacer may provide protection against the bacteriophage (or the plasmid) in potential future encounters, and/or add an extra level of regulation to the prokaryotic cell when targeting its own genome. The term“CRISPR-Cas system” may also refer to a plurality of (different) CRISPR-Cas systems, i.e., a prokaryote may comprise two or more CRISPR-Cas systems, especially wherein each CRISPR-Cas system comprises a respective CRISPR array and a respective set of Cas proteins. Typically, the CRISPR array and the genes encoding the CRISPR associated proteins may be arranged in close proximity on the genome, i.e., as part of a gene cluster (also including the CRISPR array), also referred to as a CRISPR locus. It will be clear to the person skilled in the art, however, that two separate CRISPR-Cas systems of a prokaryote may be partially overlapping in the Cas proteins involved in the respective systems.

The term“active CRISPR-Cas system” herein refers to a CRISPR-Cas system that is actively transcribed, especially wherein one or more spacers of the CRISPR array provide CRISPR immunity, more especially a CRISPR-Cas system that has an adaptive immunity function, such as a CRISPR-Cas system that acquires new spacers upon infection with a newly encountered bacteriophage (or plasmid); and/or wherein one or more spacers of the CRISPR array target specific positions in the genome providing an adaptation (evolutionary) advantageous to the prokaryotic cell ( e.g ., modification of the prokaryotic surface to evade bacteriophage predation). Hence, the term“active CRISPR-Cas system” may not refer to DNA fragments that appear to have been evolutionary derived from a CRISPR-Cas system that has lost its functionality over (evolutionary) time. For example, S. thermophilus may comprise four different CRISPR-Cas systems: CRISPRl, CRISPR2, CRISPR3, and CRISPR4, arranged at four different genomic loci, wherein CRISPRl and CRISPR3 may generally be regarded as active CRISPR-Cas systems, whereas CRISPR2 and CRISPR4 may not be actively described, and may generally be regarded as inactive CRISPR-Cas systems.

Herein, a phrase such as“a BIM comprising a spacer” will be understood as the BIM comprising a CRISPR array comprising the spacer arranged adjacent to a repeat, especially a BIM comprising an active CRISPR-Cas system comprising the CRISPR array comprising the spacer arranged adjacent to a repeat, especially such that the BIM can recognize target sequences corresponding to the spacer.

A bacteriophage insensitive mutant (BIM) of a parent strain is a strain (derived from the parent strain) that has insensitivity to a bacteriophage that does successfully infect the parent strain. The BIM may have one or more mutations relative to the parent strain, especially wherein at least some of the one or more mutations result in the insensitivity to the bacteriophage. The insensitivity of a BIM to a bacteriophage may be acquired via mutations related to a variety of mechanisms, including, among others, acquisition of a spacer targeting the bacteriophage in a CRISPR array, modification of cell surface receptors to prevent bacteriophage adsorption, binding and/or ejection, and/or production of a molecule binding the phage receptors.

The sensitivity of a (prokaryotic) strain to a bacteriophage may be evaluated using a phage exposure assay, such as a plaque assay. In such an assay, the strain (cells) may be entrapped in a matrix, in the form of a lawn, and exposed to the bacteriophage such that successful infection of the strain by the bacteriophage results in an observable strain clearing (killing), which may (typically) be observable by eye. However, if a too low concentration of bacteriophage is used for the exposure assay, it may be possible that the bacteriophage actually successfully infects but that the infection is below the limit of detection of the (plaque) assay. As a result, a strain may be considered insensitive to a bacteriophage when in reality it is infected by the phage but at such low efficiency that it is not being seen.

Hence, a strain is generally and herein considered “insensitive” to a bacteriophage when no observable strain clearing (killing) is formed on a bacterial lawn of the strain upon infection with a bacteriophage at a concentration of at least 10 8 pfu/ml in an exposure assay. The concentration of 10 8 pfu/ml allows the detection of phage infection even if the efficiency of the phage is severely reduced, such as via a resistance mechanism of the (prokaryotic) strain. The person skilled in the art will select an appropriate assay to determine insensitivity dependent on the type of bacteriophage. For example, for lytic phages, the exposure assay may typically comprise a plaque assay (see procedure below), as lytic bacteriophages may cause directly visible damage.

For lysogenic or chronic bacteriophages a similar assay may be used (as known to the person skilled in the art). If it is not possible to observe direct visible damage, the prokaryotes may be exposed to a marker allowing the selection of phage-containing bacteria.

Similarly, a strain may be considered“sensitive” to a bacteriophage when a phage plaque is formed on a bacterial lawn of the strain upon infection with a bacteriophage at a concentration of at least 10 s pfu/ml of the bacteriophage. Hence, the term “bacteriophage insensitive mutant” refers to a strain (derived from the parent strain) for which a bacterial lawn of the strain does not result in the formation of phage plaques upon infection with bacteriophages at a concentration of at least 10 s pfu/ml of the bacteriophage. An nCBIM does not rely on a CRISPR-Cas system for the insensitivity to the bacteriophage. If a BIM does not comprise a spacer targeting the bacteriophage, the BIM is an nCBIM.

A CBIM relies on a CRISPR-Cas system for the insensitivity to the bacteriophage. If a BIM comprises a spacer targeting the bacteriophage, this may be an indication that the BIM is a CBIM. It will be clear to the person skilled in the art, however, that a BIM may comprise such a spacer while (also) being insensitive to the corresponding bacteriophage via a non-CRISPR-Cas-mediated mechanism; such a BIM is herein considered an nCBIM. This may occur, for example, if an nCBIM is derived, such as derived via the method described herein, from a parent strain (cell) comprising a spacer targeting the bacteriophage.

The term“parent strain” is used herein to refer to a (bacteriophage-sensitive) strain from which a BIM may be generated. The term“parent strain cells” refers to a plurality of cells belonging to the parent strain. The term“parent strain” may herein also refer to parent strain cells. In specific embodiments, the parent strain cells may comprise Streptococcus thermophilus (cells). It will be clear to the person skilled in the art that a phrase such as“the parent strain cells comprise S. thermophilus” indicates that the parent strain cells comprise S. thermophilus cells. In general, in embodiments, only a single parent strain is used, i.e., (substantially) all prokaryotic cells used belong to the same strain. However, in further embodiments, a plurality of parent strains may be used, especially wherein at least two of the plurality of parent strains belong to different species.

The parent strain may be cultured in a growth medium suitable for the parent strain to generate biomass for the method as described herein. Growth media suitable for the parent strain will be known to the person skilled in the art. In embodiments, the method may comprise a biomass accumulation stage comprising culturing the parent strain (cells) in (or on) a growth medium suitable for the parent strain (cells).

The invention is herein, for explanatory purposes, primarily described with respect to Streptococcus thermophilus. However, it will be clear to the person skilled in the art that the invention is not limited to the generation of liCBIMs of A thermophilus. Hence, in embodiments, the parent strain cells may comprise a prokaryote, such as a bacterium and/or an archaeon, especially a lactic acid bacterium, such as a bacterium belonging to the group consisting of the genera Lactococcus, Leuconostoc, Lactobacillus , and Streptococcus , more especially a bacterium belonging to the genus Streptococcus. In specific embodiments, the parent strain cell may comprise a bacterium belonging the family Enter obacteriaceae, especially a bacterium belonging to the genus Escherichia.

In embodiments, the parent strain (cells) comprise an active CRISPR-Cas system. In further embodiments, the parent strain (cells) may comprise an (active) CRISPR- Cas system selected from the group comprising a type I CRISPR-Cas system, a type II CRISPR-Cas system, a type III CRISPR-Cas system, a type IV CRISPR-Cas system, a type V CRISPR-Cas system, a type VI CRISPR-Cas system, and combinations thereof The term “type I CRISPR-Cas system” and similar terms may also refer herein to subtypes thereof, for example to one or more of subtypes I-A, I-B, I-C, I-D, I-E, I-F, I-U and combinations thereof In further embodiments, the parent strain cells may comprise a type II CRISPR-Cas system.

Further, in embodiments, the parent strain (cells) may comprise a plurality of (active) CRISPR-Cas systems, and the anti-CRISPR protein may inhibit all of the plurality of (active) CRISPR-Cas systems. For example, S. thermophilus is presently known to comprise four CRISPR-Cas systems, of which two (CRISPR1 and CRISPR3) may be generally considered active and may be classified as type II CRISPR-Cas systems, i.e., S. thermophilus may comprise one or more (active) type II CRISPR-Cas systems. The anti- CRISPR protein corresponding to SEQ ID NO: 1 inhibits all (active) CRISPR-Cas systems of S. thermophilus. Hence, in embodiments, the anti-CRISPR protein may be suitable to inhibit type II CRISPR-Cas systems, especially a plurality of type II CRISPR-Cas systems, such as CRISPRl and CRISPR3 of S. thermophilus. However, as will be clear to the person skilled in the art, the present invention is not limited to parent strain (cells) that comprise (only) type II CRISPR-Cas systems as (i) ACRs are known for other types of CRISPR-Cas systems, (ii) an ACR may inhibit multiple types of CRISPR-Cas systems, and (iii) the parent strain cells may be (simultaneously) exposed to multiple different ACRs configured to inhibit a plurality of CRISPR-Cas systems.

An anti-CRISPR protein (also“ACR” or“Acr”) is a protein configured to inhibit the activity of a CRISPR-Cas system. Typically, ACRs may be of viral origin. ACRs may employ various mechanisms to inhibit the activity of a CRISPR-Cas system, such as covering a DNA recognition groove, preventing the binding to target DNA sequences, directly interacting with the catalytic residues of a nuclease domain of a Cas protein, and/or by enzymatic activity on the guide’s end (crRNA) or the nuclease residues. The term“anti- CRISPR protein” may also refer to a plurality of different anti-CRISPR proteins, especially different anti-CRISPR proteins suitable to inhibit different CRISPR-Cas systems (see further below). Hence, in embodiments, the anti-CRISPR protein exposure stage may comprise exposing the parent strain cells to two or more different anti-CRISPR proteins, especially wherein the two or more different anti-CRISPR proteins are at least partially non overlapping in with respect to the CRISPR-Cas systems they (the two or more anti-CRISPR proteins) inhibit.

The term“stage” used herein refers to a (time) period (also“phase”) of the method. The different stages may (partially) overlap (in time). For example, the anti- CRISPR protein exposure stage may, in general, be initiated prior to the bacteriophage exposure stage, and may continue until the end of the bacteriophage exposure stage or even longer. It will be clear to the person skilled in the art how the stages may be beneficially arranged in time. For example, the anti-CRISPR protein exposure stage may last during substantially all of the bacteriophage exposure stage, and the selection stage may typically occur after the bacteriophage exposure stage. Hence, in specific embodiments, the anti- CRISPR protein exposure stage may be initiated, especially arranged, prior to the bacteriophage exposure stage. In further embodiments, the selection stage may be arranged after the bacteriophage exposure stage.

The anti-CRISPR protein exposure stage may comprise exposing the parent strain cells to an anti-CRISPR protein, especially an anti-CRISPR protein suitable for inhibiting the CRISPR-Cas system, more especially for inhibiting spacer acquisition by the (inhibited) CRISPR-Cas system. In embodiments, the anti-CRISPR protein exposure stage may comprise exposing the parent strain cells to an anti-CRISPR protein at a concentration sufficient to inhibit the CRISPR-Cas system. Especially, the anti-CRISPR protein exposure stage may comprise exposing the parent strain cells to an anti-CRISPR protein at a concentration sufficient to inhibit > 90% of the activity of the CRISPR-Cas system, such as > 95%, especially > 97%, such as > 98%, especially > 99%, including up to 100%. More especially, the anti-CRISPR protein exposure stage may comprise exposing the parent strain cells to an anti-CRISPR protein at a concentration sufficient to inhibit > 90% of CRISPR spacer acquisition by the inhibited CRISPR-Cas system, such as > 95%, especially > 97%, such as > 98%, especially > 99%, including up to 100%. It will be clear to the person skilled in the art that the concentration of anti-CRISPR protein a parent strain may need to be exposed to for a desired inhibition percentage may be dependent on, for example, the parent strain and the anti-CRISPR protein, i.e., different concentrations may be used for different parent strains and/or different anti-CRISPR proteins. The activity of a CRISPR-Cas system may be determined via a plasmid transformation assay as described in Almendros and Mojica 2015,“Exploring CRISPR Interference by Transformation with Plasmid Mixtures: Identification of Target Interference Motifs in Escherichia coif , which is hereby incorporated by reference. Essentially, CRISPR interference (immunity) may be estimated by the efficiency of transforming a prokaryote with a high-copy number plasmid exposed to the marker gene (selective pressure, e.g antibiotic resistance gene). After cultivation, the number of cells (colonies) may be indirectly proportional to the CRISPR activity. Alternatively, the activity of a CRISPR-Cas system may be determined by efficiency of plating/plaquing (pfu/ml) as described in Barrangou et al 2007“CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes”, which is herein incorporated by reference. Here, CRISPR immunity may be estimated by the number of plaques formed on a bacterial lawn comprising a prokaryote comprising one or more spacers targeting the bacteriophage of interest. As before, pfu may be indirectly proportional to CRISPR activity. Hence, both assays may inform a person skilled in the art with regards to DNA recognition and cleavage. Specifically, for the purposes of anti-CRISPR inhibition, strains exposed to different concentrations of anti- CRISPR protein may be subjected to aforementioned assays to identifiy a suitable amount of anti-CRISPR protein for CRISPR inhibition, such as an amount of anti-CRISPR protein sufficient to inhibit the CRISPR-Cas system, especially sufficient to inhibit > 90% of the activity of the CRISPR-Cas system.

In embodiments, the anti-CRISPR protein exposure stage may comprise exposing the parent strain cells to the anti-CRISPR protein in an internal copy number equal to or larger than the number of (targeted) CRISPR-Cas proteins (in a cell). For example, if the anti-CRISPR protein specifically targets a CRISPR-Cas nuclease, the internal copy number of the anti-CRISPR protein may be equal to or larger than the number of CRISPR- Cas nuclease proteins (in a cell ). The term“internal copy number” refers to the number of anti-CRISPR proteins inside an individual (parent strain) cell. For example, a specific individual (parent strain) cell may comprise 10-40 CRISPR-Cas proteins, but depending on the species and the (growth) conditions a cell may also have fewer or more CRISPR-Cas protein. Hence, in specific embodiments, the anti-CRISPR protein exposure stage may comprise exposing the parent strain cells to the anti-CRISPR protein in an internal copy number of at least 10 of the anti-CRISPR protein, such as at least 50, especially at least 100, such as at least 500, especially at least 1000, such as at least 5000. In further embodiments, the anti-CRISPR protein exposure stage may comprise exposing the parent strain cells to the anti-CRISPR protein in an internal copy number of at most 10000 of the anti-CRISPR protein, such as at most 5000, especially at most 1000, such as at most 500. In further embodiments, the anti-CRISPR protein exposure stage may comprise exposing the parent strain cells to the anti-CRISPR protein in a concentration suitable to provide the internal copy number.

In embodiments, the anti-CRISPR protein exposure stage may comprise exposing the parent strain cells to the anti-CRISPR protein in a concentration of at least 0.1 mM of the anti-CRISPR protein, especially 1 mM, such as at least 2 miM, especially at least 5 pM, such as at least 10 miM, especially at least 20 pM, such as at least 50 pM, especially at least 100 pM. In further embodiments, the anti-CRISPR protein exposure stage may comprise exposing the parent strain cells to the anti-CRISPR protein in a concentration of at most 100 niM of the anti-CRISPR protein, such as at most 10 mM, especially at most 1 niM, such as at most 100 pM, especially at most 50 miM, such as at most 20 miM.

In embodiments, the parent strain cells may comprise a plurality of (active) CRISPR systems, and the anti-CRISPR protein may be suitable for inhibiting all of the plurality of (active) CRISPR systems, especially for inhibiting spacer acquisition by the (inhibited) CRISPR-Cas systems. In further embodiments, the parent strain cells may comprise S. thermophilus, and the anti-CRISPR protein may comprise a protein with the amino acid sequence SEQ ID NO: 1. In such embodiment, the anti-CRISPR protein may be suitable to inhibit both the CRISPR 1 and CRISPR3 loci of S. thermophilus.

In embodiments, the anti-CRISPR protein may comprise a protein with the amino acid sequence SEQ ID NO: l. In further embodiments, the anti-CRISPR protein may comprise a protein with an amino acid sequence different from SEQ ID NO: 1, especially an amino acid sequence similar to SEQ ID NO: 1. In general, if two proteins consist of (highly) similar amino acid sequences, these two proteins may be likely to perform the same biological function. This relation between amino acid sequence and protein function may, for example, be used to predict the function of a protein based on its sequence identity with proteins of known function (annotation by sequence homology based inference). The term “sequence identity” herein refers to the percentage of the characters (such as amino acids in a protein sequence) in the shorter of two sequences matching an identical character in the longer of the two sequences in a sequence alignment (also see below). The higher the sequence identity between two proteins, the higher the chance may be that these two proteins have the same or a similar function. Although there may not be a hard rule for inferring functional identity or similarity based on a threshold value for sequence identity, especially as the threshold value may need to be adjusted based on (relative) sequence length and/or protein function, proteins may have been successfully annotated based on a common rule- of-thumb threshold of at least 30-40% sequence identity. Hence, proteins similar to SEQ ID NO: l in both length (including both shorter and longer) and amino acid sequence may have a similar anti-CRISPR activitity.

Hence, in embodiments, the anti-CRISPR protein may comprise a protein comprising, especially consisting of, an amino acid sequence, wherein the amino acid sequence has at least 30% sequence identity with the amino acid sequence of SEQ ID NO: 1, such as at least > 40% sequence identity, especially > 50%, such as > 60%, especially > 70%, such as > 75%, especially > 80%, such as > 85%, especially > 90%, such as > 93%, especially > 95%, such as > 97%, including 100% sequence identity, with respect to a sequence alignment between the amino acid sequence and SEQ ID NO: l, wherein the sequence alignment has a length > 50% of the sequence length of SEQ ID NO: 1, such as > 60%, especially > 70%, such as > 80, especially > 90, such as > 99%, including 100%, wherein the protein has anti-CRISPR properties similar to SEQ ID NO: 1.

Hence, in further embodiments, the amino acid sequence may be shorter or longer than SEQ ID NO: 1. Hence, in embodiments, the anti-CRISPR protein may comprise a protein comprising, especially consisting of, an amino acid sequence, wherein the amino sequence has a sequence length > 50% of the sequence length of SEQ ID NO: 1, such as > 60%, especially > 70%, such as > 80, especially > 90, such as > 100%, especially > 120%, wherein a protein comprising the amino acid sequence has anti-CRISPR properties similar to SEQ ID NO: l . Similarly, in further embodiments, the amino acid sequence may have a sequence length < 200% of the sequence length of SEQ ID NO: 1, such as < 180%, especially

< 160%, such as < 140%, especially < 130%, such as < 120%, especially < 1 10%, such as

< 100%, wherein a protein comprising the amino acid sequence has anti-CRISPR properties similar to SEQ ID NO: 1.

In further embodiments, the anti-CRISPR protein may comprise a multimer of anti-CRISPR proteins, such as a dimer of anti-CRISPR proteins. In specific embodiments, the anti-CRISPR protein may comprise a homomultimer, especially a homodimer, of an anti-CRISPR protein, especially of an anti-CRISPR protein with an amino acid sequence. In further specific embodiments, the anti-CRISPR protein may comprise a heteromultimer, especially a heterodimer, of different anti-CRISPR proteins, especially wherein at least two of the different anti-CRISPR proteins comprise a (different) amino acid sequence. A comparison of sequence identity between two or more amino acid sequences may generally be performed with regards to the amino acid sequences prior to any potential post-translational modifications. Hence, the amino acid sequences may be compared as they are genetically encoded in their corresponding genes, especially translated via the appropriate codon table, such as generally via the canonical codon table.

A sequence alignment of two (or more) sequences may be obtained using, for example, BLASTp. BLASTp is a Basic Local Alignment Search Tool for proteins and will be familiar to the person skilled in the art. BLASTp may be used to align a query amino acid sequence against an other amino acid sequence, especially with default settings, and may provide an alignment indicating the“query coverage” of the query amino acid sequence(s) (the percentage of the query amino acid sequence successfully aligned with the other amino acid sequence) and a sequence identity. The phrase“a sequence alignment having a sequence length > 50% of the sequence length of SEQ ID NO: 1” will be understood to refer to a“query coverage” of > 50% for a BLASTp alignment with SEQ ID NO: 1 as the query amino acid sequence.

For example, a BLASTp with SEQ ID NO: l as query sequence against the non-redundant protein database on 25 January 2019 with default settings except for number of possible hits (1000 instead of 100) yielded 107 hits, 105 of which have a query coverage > 50% and a sequence identity > 30%, 96 of which have a query coverage > 75% and a sequence identity > 70%. Hence, in embodiments the anti-CRISPR protein may comprise an amino acid sequence corresponding to an accession number selected from the group consisting of NP_049960.1, NP_597804.1, AZF92186.1, ATI17220.1, YP_008772077.1, ATI19545.1, ARU14225.1, AZF90597. 1, AZF90839.1, AZF91934.1, AYP30067.1, AYP29070.1, NP_695096.1, AZF90493.1, AZF90386.1, AZF90647.1, YP_007392266.1, AZF90929.1, ARU13794.1, AZF91980.1, AYP29666.1, AYP29576.1, ARU14275.1, ETW90629.1, YP_238524.1, ARU13392.1, AUN43350.1, ALJ99658.1, AYP29621.1, NP_038313.1, AZF91500.1, AZF92771.1, AYP29245.1, ARU13566.1, ARU13524.1, AZF91558.1, ASD51009.1, AZF90749.1, ARU14640.1, AZF92131.1, AZF92330.1, ALJ99574.1, ARU14732.1, APC45905.1, NP_049428.1, ARU13933.1, APC45860.1, ARU14686.1, AZF90696.1, AYP29489.1, AYP29445.1, YP_009286869.1, ARU13438.1, AYP28665.1, AZF92374.1, ARU13287.1, AZF91130.1, YP_009280368.1,

YP_002925091.1, APC45814.1, AZF90979.1, AZF92420.1, ATI19629. 1, ATI19707.1, ARU13341.1, ATI19922.1, ARU14549.1, ARU13197.1, AYP29109.1, YP_003347448.1, AYP29366.1, ARU14597.1, AYP29200.1, ARU13095.1, AYP29935.1, AUN43359.1, ARU12994.1, ARU13041.1, ATI17177.1, AZF90443.1, AZF91407.1, AUN43428.1, ALJ99619.1, AZA24392.1, ARU14036.1, AZF92689.1, ATP9588.1, AYP30313.1, ARU13985.1, YP_009003363.1, AZF89036.1, AZF91222.1, SHM57908.1,

YP_006561251.1, ASN68204.1, AZF92856.1, AQP41496.1, SNI15902.1, EHJ56856.1, SQG83716.1, CBI12938.1, WP_012679305.1, WP_050316385.1, WP_050321512.1, VEB81257.1.

SEQ ID NO: 1 may have a 100% identity with the sequence corresponding to NCBI accession number NP 049960.1 with a query coverage of 100%. Hence, in embodiments, the anti-CRISPR protein comprises a protein comprising an amino acid sequence, wherein the amino acid sequence has at least 30% sequence identity with the amino acid sequence corresponding to NCBI accession number NP 049960.1 with respect to a sequence alignment between the amino acid sequence and amino acid sequence corresponding to NCBI accession number NP 049960.1, and wherein the sequence alignment has a length > 50% of the sequence length of amino acid sequence corresponding to NCBI accession number NP 049960.1.

In embodiments, the parent strain cells may express a gene encoding the anti- CRISPR protein, i.e., the anti-CRISPR protein exposure stage may comprise the parent strain cells producing the anti-CRISPR protein.

In further embodiments, the gene may encode an amino acid sequence, wherein the amino acid sequence has at least 30% sequence identity with the amino acid sequence of SEQ ID NO: 1, such as at least > 40% sequence identity, especially > 50%, such as > 60%, especially > 70%, such as > 75%, especially > 80%, such as > 85%, especially > 90%, such as > 93%, especially > 95%, such as > 97%, including 100% sequence identity, with respect to a sequence alignment between the amino acid sequence and SEQ ID NO: 1, wherein the sequence alignment has a length > 50% of the sequence length of SEQ ID NO: 1, such as > 60%, especially > 70%, such as > 80, especially > 90, such as > 99%, including 100%, wherein the protein has anti-CRISPR properties similar to SEQ ID NO: 1. Hence, in a specific embodiment, the amino acid sequence may, for example, have a sequence length equal to 96% of the sequence length of SEQ ID NO: 1, and may have a sequence identity of 84% with SEQ ID NO: 1.

In further embodiments, the parent strain cells may comprise a plasmid, wherein the plasmid comprises the gene, wherein the gene encodes the anti-CRISPR protein. In further embodiments, the parent strain cells may constitutively express the gene (encoding the anti-CRISPR protein).

In further embodiments, the parent strain cells may be configured to express the gene (encoding the anti-CRISPR protein) when induced, i.e., the gene (encoding the anti-CRISPR protein) may comprise an inducible gene. In such embodiment, the anti- CRISPR protein exposure stage may comprise inducing the expression of the inducible gene. In further embodiments, the inducible gene may comprise a repressed gene and the anti-CRISPR protein stage may comprise relieving the repression of the repressed gene.

In further embodiments, the parent strain cells may express a gene, wherein the gene encodes the anti-CRISPR protein, and wherein the gene is located on the genome of the parent strain cells, i.e., the parent strain genome comprises the gene. Hence, in embodiments, the parent strain genome may be genetically modified to comprise the gene. Methods for modifying a bacterial genome are known to the person skilled in the art.

The bacteriophage exposure stage may comprise exposing the parent strain cells to a bacteriophage, especially a bacteriophage capable of infecting (at least some of) the parent strain cells. The method of the invention may essentially be applicable for any bacteriophage capable of infecting the parent strain cells. In specific embodiments, the bacteriophage may comprise one or more bacteriophages selected from the group (of bacteriophages) recognized by the International Committee on Taxonomy of Viruses (ICTV) able to infect the bacterial cell, such as one or more bacteriophages selected from the group (of bacteriophages) mentioned in the 9 th ICTV report on Virus Taxonomy (201 1), such as the order Caudovirales. Hence, in embodiments, the bacteriophage may comprise one or more bacteriophages selected from the order Caudovirales.

In embodiments, the bacteriophage exposure stage may comprise exposing the parent strain cells to a bacteriophage in a suitable concentration, which may vary depending on the phage and/or the parent strain as known to the person skilled in the art. In specific embodiments, the bacteriophage exposure stage may comprise exposing the parent strain cells to the bacteriophage in a concentration > 10 1 pfu/ml of the bacteriophage, such as > 10 2 pfu/ml, especially > 10 3 pfu/ml, such as > 10 4 pfu/ml, especially 1CP pfu/ml, such as > 10 6 pfu/ml, such as > 10 7 pfu/ml, especially > 10 s pfu/ml.

The term “bacteriophage” may also refer to a plurality of different bacteriophages, i.e., genetically distinct bacteriophages. Hence, in embodiments, the bacteriophage exposure stage may comprise exposing the parent strain cells to a plurality of different bacteriophages, especially a plurality of bacteriophages (each) capable of infecting (at least some of) the parent strain cells.

In embodiments, the bacteriophage exposure stage may comprise one or more passages, especially wherein each passage comprises a bacteriophage exposure, i.e., exposing the parent strain cells to the bacteriophage. A passage may comprise: growing the parent strain cells to a desired cell density in a growth medium, especially in a liquid culture; (then) exposing the parent strain cells to the bacteriophage; (then) transferring at least part of surviving bacterial (parent strain) cells to a new growth medium. If a second passage follows the passage, the new growth medium may be suitable for the second passage, for example, a new liquid culture. If the passage was the last passage, the new growth medium may be suitable for the (optional) selection stage; for example, the new growth medium may be an agar plate. In embodiments, the bacteriophage exposure stage may comprise n passages, wherein n > 1, especially n > 2, such as n > 3, especially n > 5, such as n > 7, especially n > 10, such as n > 15. In further embodiments, n < 25, such as < 20, especially < 15, such as < 10.

In embodiments, the bacteriophage exposure stage may comprise exposing the parent strain (cells) to the bacteriophage for a time period. In general, the time period may be selected such that the bacteriophage has had sufficient time to infect essentially all of the parent strain cells, which may vary for different parent strain cells and/or bacteriophages. In further embodiments, the bacteriophage exposure stage, especially a passage, more especially each passage, may comprise exposing the parent strain cells to the bacteriophage for a time period of least 4 hours, such as at least 6 hours, especially at least 8 hours, such as at least 10 hours, especially at least 12 hours. In further embodiments, the time period may be at most 24 hours, such as at most 20 hours, especially at most 16 hours, such as at most 12 hours, especially at most 10 hours. In specific embodiments, the time period may be selected from the range of 8 to 12 hours.

In specific embodiments, the bacteriophage exposure stage may comprise n passages, wherein each passage comprises exposing the parent strain cells to the bacteriophage for a time period selected from the range of 4 - 24 hours, especially from the range of 8 to 12 hours.

In further embodiments, the bacteriophage exposure stage may comprise exposing the parent strain to a plurality of bacteriophages. Especially, at least one of the passages, more especially each passage, may comprise exposing the parent strain to a plurality of different bacteriophages. Simultaneously exposing the parent strain to a plurality of different bacteriophages may be beneficial as two or more bacteriophages may recombine to provide a hybrid bacteriophage, which may pose further challenges to the parent strain. By allowing for hybrid bacteriophage formation during the bacteriophage exposure stage, the resulting nCBIM may be more robust towards hybrid bacteriophages that may be encountered during application of the nCBIM, for example in an industrial setting. The use of the ACR may provide increased hybrid bacteriophage generation as a bacteriophage that would otherwise be targeted by the CRISPR-Cas system may avoid CRISPR-Cas facilitated degradation, which may result in increased hybrid bacteriophage generation involving this particular bacteriophage.

In embodiments, the anti-CRISPR protein exposure stage may further comprise exposing the parent strain cells to an auxiliary inhibitor. In further embodiments, the auxiliary inhibitor may comprise one or more of an antisense RNA, a second anti- CRISPR protein, and an anti-CRISPR-associated protein, especially one or more of an second anti-CRISPR protein, and an anti-CRISPR-associated protein, more especially an anti-CRISPR-associated protein, or especially one or more of an antisense RNA and an anti- CRISPR-associated protein, more especially an antisense RNA. The parent strain cells may be exposed to the auxiliary inhibitor prior to, simultaneously with, or subsequently to the exposure to the anti-CRISPR protein. In general, if an auxiliary inhibitor is used, the anti- CRISPR protein exposure stage may comprise exposing the parent strain cells to the anti- CRISPR protein and the auxiliary inhibitor simultaneously.

In embodiments, the bacteriophage may comprise a gene encoding the anti- CRISPR protein. In general, however, the anti-CRISPR protein may be provided to the parent strain cells independent of the bacteriophage, i.e., the parent strain cells are exposed to the anti-CRISPR protein prior to being exposed to the bacteriophage.

In embodiments, the anti-CRISPR protein exposure stage may comprise providing the anti-CRISPR protein to the parent strain cells via direct protein addition. In such embodiments, the parent strain cells may take up the anti-CRISPR protein from their environment. In particular, the parent strain cells, especially membranes of the parent strain cells, may be made more permeable to facilitate the uptake of the anti-CRISPR protein. Hence, in further embodiments, the anti-CRISPR protein exposure stage may comprise one or more of providing a cell-penetrating peptide, cell squeezing, and electroporation (to the parent strain (cells)). This may facilitate introduction of the anti-CRISPR protein into the parent strain cells. Hence, in embodiments, the anti-CRISPR protein exposure stage may comprise exposing the parent strain cells to a cell-penetrating peptide. The cell-penetrating peptide may be configured to increase the uptake of the anti-CRISPR protein by the parent strain cells. In further embodiments, the cell-penetrating peptide may be configured to associate with the anti-CRISPR protein, especially via non-covalent interactions, or especially via covalent interactions.

In embodiments, the anti-CRISPR protein exposure stage may comprise exposing the parent strain (cells) to an RNA molecule encoding the anti-CRISPR protein. In such embodiments, the parent strain cells may take the RNA molecule up from their environment. In particular, the RNA molecule may be delivered into the parent strain cells via transfection, especially via one or more of microinjection, electroporation, calcium co precipitation, cationic polymers and lipids, cell-penetrating peptides and cell-penetrating particles.

In embodiments, the method may further comprise a selection stage. The selection stage may comprise selecting the bacteriophage insensitive mutant from (surviving) cells having survived the bacteriophage exposure stage. The selection stage may comprise growing the cells having survived the bacteriophage exposure (also“surviving cells)” on a plate, such as on an agar plate. In embodiments, the selection stage may further comprise assessing characteristics, especially one or more of phenotypic and/or genetic features, of the cells having survived the bacteriophage exposure stage, such as assessing characteristics of colonies on the plate.

Hence, in embodiments, the selection stage may comprise assessing phenotypic features of the cells having survived the bacteriophage exposure stage, especially assessing phenotypic features, such as phage resistance, of colonies on a plate.

In further embodiments, the selection stage may comprise assessing genetic features of the cells having survived the bacteriophage exposure stage, especially assessing genetic features of colonies on the plate. For example, the selection stage may comprise determining one or more CRISPR arrays of the cells having survived the bacteriophage exposure stage.

In embodiments, the selection stage may further comprise comparing CRISPR arrays of the surviving cells, especially cells derived from the surviving cells, with CRISPR arrays of the parent strain cells and selecting the bacteriophage insensitive mutant (from the surviving cells) based on having an identical CRISPR array to the CRISPR array of the parent strain cells, i.e., the spacers in the CRISPR array of the BGM are identical (have an identical sequence) to the spacers in the CRISPR array of the parent strain cells.

In embodiments wherein the parent strain cells comprise a plasmid, wherein the plasmid comprises the gene, wherein the gene encodes the anti-CRISPR protein, the method may further comprise curing the surviving cells, especially cells derived from the surviving cells, of the plasmid. In further embodiments, the method may comprise curing the BIM of the plasmid.

In embodiments, the method may comprise generating a non-GMO nCBIM. Hence, in embodiments the BIM may comprise a non-GMO non-CRISPR-Cas-mediated bacteriophage insensitive mutant.

In general, the method may be executed in a laboratory and/or industrial setting. However, in specific embodiments, the method may be performed in a non laboratory non-industrial setting. For example, the anti-CRISPR protein may be provided to a (natural) environment comprising prokaryotes and bacteriophages, especially such that the prokaryotes develop a non-CRISPR-based resistance to the bacteriophages. In specific embodiments, the method may comprise providing the anti-CRISPR protein to the gut microbiota of a subject. Thereby, the method may aid in developing a microbiota more robust towards bacteriophages.

In embodiments, the method may be a non-medical method.

In a second aspect, the invention further provides a (non-CRISPR-Cas- mediated) bacteriophage insensitive mutant obtained using the method as described herein. In embodiments, the BIM may comprise a prokaryote, such as a bacterium and/or an archaeon, especially a lactic acid bacterium, such as a bacterium belonging to the group consisting of the genera Lactococcus, Leuconostoc, Lactobacillus , Escherichia and Streptococcus , more especially Streptococcus thermophilu . Hence, in embodiments, the BIM may be obtained using the method according to the invention with parent strain cells comprising Streptococcus thermophilus.

In yet a further aspect, the invention further provides a (non-CRISPR-Cas- mediated) bacteriophage insensitive mutant obtainable by the method as described herein.

In embodiments, the (non-CRISPR-Cas-mediated) bacteriophage insensitive mutant may have a reduced sensitivity to one or more bacteriophages selected from the group recognized by the International Committee on Taxonomy of Viruses (ICTV) able to infect the bacterial cell, especially one or more bacteriophages selected from the order Caudovirales , especially, the BIM may be insensitive to one or more bacteriophages selected from the group recognized by the International Committee on Taxonomy of Viruses (ICTV) able to infect the bacterial cell, especially one or more bacteriophages selected from the order Caudovirales, such as not forming a plaque in a plaque assay when exposure to lO 8 pfu/ml of the one or more bacteriophages.

In a further aspect, the invention provides a use of the (non-CRISPR- mediated) bacteriophage insensitive mutant according to the invention, especially a BIM obtained with the method according to the invention, in a process for producing a product, especially a food product, or especially a dairy product, or especially a probiotic product. The use of the nCBIM in a production process may be beneficial as the production is less likely to be hampered by a bacteriophage infection compared to a production process using the parent strain.

In specific embodiments, the use may comprise producing a food product, especially a dairy product. Prokaryotes used during food production may end up in the food product. For example, yoghurt may generally comprise the lactic acid bacteria that were used to produce the yoghurt. In further embodiments, the food products, especially the dairy product, may comprise a probiotic (product), especially the bacteriophage insensitive mutant according to the invention. In further embodiments, the food product may comprise a dairy product.

Hence, in a further aspect, the invention provides a food product comprising the (non-CRISPR-mediated) bacteriophage insensitive mutant according to the invention, especially comprising a BIM obtained with the method according to the invention.

In a further aspect, the invention provides a dairy product comprising the (non-CRISPR-mediated) bacteriophage insensitive mutant according to the invention, especially comprising a BIM obtained with the method according to the invention.

In a further aspect, the invention may provide a probiotic (product) comprising the (non-CRISPR-mediated) bacteriophage insensitive mutant according to the invention.

In a further aspect, the invention further provides a composition comprising an anti-CRISPR protein, wherein the anti-CRISPR protein comprises at least 30% sequence identity with the amino acid sequence of SEQ ID NO: 1. In embodiments, the composition may comprise an auxiliary inhibitor.

In a further aspect, the invention further provides a polynucleotide comprising a sequence encoding an anti-CRISPR protein, wherein the anti-CRISPR protein comprises at least 30% sequence identity with the amino acid sequence of SEQ ID NO: l, wherein the polynucleotide is configured to be provided to a cell for expression.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which:

Fig. l schematically depicts an embodiment of the method 100 for the generation of a bacteriophage insensitive mutant 250.

The schematic drawings are not necessarily on scale.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Fig. 1 schematically depicts an embodiment of the method 100 for the generation of a bacteriophage insensitive mutant 250 from parent strain cells 200 comprising an active CRISPR-Cas system. In the depicted embodiment, a liquid cell culture comprises the parent strain cells 200. The method 100 comprises the stages: anti-CRISPR protein exposure stage 110, bacteriophage exposure stage 120, selection stage 130.

Prior to the anti-CRISPR protein exposure stage 110, the parent strain cells 200 may be grown to a desired cell density. In the depicted embodiment, the parent strain cells 200 were grown in a liquid culture in an erlenmeyer flask, for example to an OD600 of 0.2.

The anti-CRISPR protein exposure stage 110 comprises exposing the parent strain cells 200 to an anti-CRISPR protein 1 15 suitable for inhibiting the CRISPR-Cas system. In the depicted embodiment, the anti-CRISPR protein exposure stage 1 10 comprises providing the anti-CRISPR protein 115 to the parent strain 200 cells via direct protein addition. In embodiments, the anti-CRISPR protein exposure stage 110 may comprise exposing the parent strain cells 200 to the anti-CRISPR protein 1 15 in a concentration of at least 10 mM of the anti-CRISPR protein. In yet further embodiments, the anti-CRISPR protein exposure stage 1 10 may further comprise exposing the parent strain cells 200 to an auxiliary inhibitor, especially wherein the auxiliary inhibitor comprises one or more of an antisense RNA, a second anti-CRISPR protein, and an anti-CRISPR-associated protein. In further embodiments, the anti-CRISPR protein exposure stage 110 may further comprise exposing the parent strain cells 200 to a cell-penetrating peptide. In yet further embodiments, the parent strain cells 200 may produce the anti- CRISPR protein 115, for example in embodiments wherein the parent strain cells 200 comprise a plasmid, wherein the plasmid comprises a gene, wherein the gene encodes the anti-CRISPR protein.

In embodiments, the anti-CRISPR protein 115 may comprise a protein comprising an amino acid sequence, wherein the amino acid sequence has at least 30% sequence identity with the amino acid sequence of SEQ ID NO: 1 with respect to a sequence alignment between the amino acid sequence and SEQ ID NO: 1, and wherein the sequence alignment has a length > 50% of the sequence length of SEQ ID NO: 1.

The bacteriophage exposure stage 120 comprises exposing the parent strain cells 200 to a bacteriophage 125 especially a bacteriophage 125 capable of infecting (at least some of) the parent strain cells 200. In the depicted embodiment, the bacteriophage 125 is directly added to the liquid culture of parent strain cells 200, which were previously exposed to the anti-CRISPR protein 115. In embodiments, the bacteriophage exposure stage 120 may comprise exposing the parent strain cells 200 to the bacteriophage 125 in a concentration of at least 10 1 pfu/ml of the bacteriophage. In further embodiments, the bacteriophage 125 may comprise one or more bacteriophages 125 selected from the group recognized by the International Committee on Taxonomy of Viruses (ICTV) able to infect the bacterial cell, especially one or more bacteriophages 125 selected from the group consisting of Myoviridae, Siphoviridae and Podoviridae. In further embodiments, the bacteriophage 125 may comprise one or more bacteriophages selected from the order Catidovi rales.

The bacteriophage exposure stage 120 may comprise exposing the parent strain cells 200 to the bacteriophage 125 for a time period selected such that the bacteriophage 125 has had sufficient time to infect essentially all of the parent strain cells 200, such as a time period of at least 12 hours. Hence, at the end of the bacteriophage exposure stage 120, a substantial fraction of the parent strain cells 200 may have died as a result of the exposure to the bacteriophage 125, and surviving cells (if any) may be likely to be BIMs 250, especially nCBIMs. In further embodiments, the bacteriophage exposure stage 120 may comprise one or more passages, especially wherein a passage comprises a bacteriophage exposure, i.e., exposing the parent strain cells 200 to the bacteriophage 125. In further embodiments, each passage of the one or more passages may comprise exposing the parent strain cells 200 to the bacteriophage 125 for a time period of least 4 hours, such as at least 6 hours, especially at least 8 hours, such as at least 10 hours, especially at least 12 hours. In further embodiments, each passage of the one or more passages may comprise exposing the parent strain cells 200 to the bacteriophage 125 the time period of at most 24 hours, such as at most 20 hours, especially at most 16 hours, such as at most 12 hours, especially at most 10 hours. In specific embodiments, the time period may be selected from the range of 8 to 12 hours.

In the depicted embodiment, the method 100 further comprises the selection stage 130. The selection stage 130 comprises selecting the bacteriophage insensitive mutant 250 from cells having survived the bacteriophage exposure stage 120. In the depicted embodiment, the surviving cells 201 are plated after the bacteriophage exposure stage 120. For example, after the bacteriophage exposure stage 120, the liquid culture may be spun down, washed, diluted, and then plated onto one or more agar plates to obtain colonies of surviving cells 201. In the depicted embodiment, multiple colonies of surviving cells 201 are selected and individually plated on separate agar plates and grown into bacterial lawns 210. The bacterial lawns (once fully grown) are exposed to the bacteriophage 125 at a concentration of at least 10 8 pfu/ml to determine whether or not the bacterial lawns 210 are resistant to the bacteriophage 125. The agar plates that do not form one or more phage plaques 215 after exposure to the bacteriophage 125 comprise a bacteriophage insensitive mutant 250, especially a non-CRISPR-mediated bacteriophage insensitive mutant.

In further embodiments, the selection stage may further comprise comparing CRISPR arrays of the surviving cells 201 with CRISPR arrays of the parent strain cells 200 and selecting the bacteriophage insensitive mutant 250 based on having an identical CRISPR array to the CRISPR array of the parent strain cells 200.

In embodiments wherein the parent strain cells 200 comprise a plasmid, the method 100, especially the selection stage 130, may further comprise curing the surviving cells 201 of the plasmid.

Materials and Methods

Procedure 1 : Identification of SEQ ID NO: l. The anti-CRISPR protein corresponding to SEQ ID NO: l was discovered using an in vivo three-plasmid screening system for proteins interacting with the type II-A CRISPR-Cas system. The screening was performed with E. coli (transformed with the three plasmids). Plasmid 1 expresses a red fluorescent protein (RFP) under control of a rhamnose promoter. Plasmid 2 expresses dCas9 and a sgRNA targeting the rhamnose promoter of the RFP of Plasmid 1; activity of dCas9 thus results in repression of RFP expression. Plasmid 3 contains a viral library with potential anti-CRISPR activity. If an anti-CRISPR protein affecting target recognition by dCas9 is encoded on the library, inhibition of RFP expression is blocked and RFP can be detected, e.g. by fluorescence-activated cell sorting (FACS). The viral libraries of Plasmid 3 were obtained from DNA isolated from a collection of bacteriophages infecting S. thermophilus. Bacteriophage DNA was isolated using common phenol/chloroform procedures. The bacteriophage DNA was fragmented; DNA adapters were ligated onto both sides of the fragments and the final products were PCR amplified. The amplified phage DNA fragments were cloned into the TOPO XL backbone to create the library of Plasmid 3. Screening of the library using the in vivo system led to the identification of the anti-CRISPR protein by an increase of fluorescent intensity. Interference assays using Plasmid 3 containing the anti- CRISPR protein demonstrated a 105-fold increase of recovered colonies compared to the empty plasmid, confirming the inhibition of the S. thermophilus CRISPR system. The anti- CRISPR protein 1 15 inhibits both CRISPRl and CRISPR3 of S. thermophilus. The anti- CRISPR protein 115 corresponding to SEQ ID NO: l was found to have an identical sequence to a protein sequence from Streptococcus phage P7573, specifically with hypothetical protein P7573 44 from Streptococcus phage P7573 (GenBank accession number ARU 13887.1).

Procedure 2: Genetic engineering. Plasmid transformation and genome editing techniques are known to the person skilled in the art, with many suitable reference materials. The person skilled in the art could, for example, consult Somkuti and Steinberg, 1988,“Genetic transformation of Streptococcus thermophilus” for transformation, and Ran et ah, 2013,“Genome engineering using the CRISPR-Cas9 system” for genome editing, which are hereby herein incorporated by reference.

Procedure 3 : Bacteriophage exposure. The parent strain (cells) may be grown in liquid culture until an optical density at 600 nm of 0.2. The parent strain (cells) may then be exposed to the bacteriophage at a concentration equal to or higher than 10 1 pfu/ml and incubated at a suitable incubation temperature, such as 37 °C for S. thermophilus , especially with rocking.

Procedure 4: Assessing sensitivity via a plaque assay. As known to the person skilled in the art, a plaque assay may be used to determine whether a prokaryote is insensitive to a bacteriophage. A plaque assay may, generally, comprise (i) growing the prokaryote on an (agar) plate such that the top of the plate is substantially covered in bacterial colonies, he., the bacterial colonies may have merged into a bacterial lawn, (ii) exposing the bacterial lawn to the bacteriophage, especially to the bacteriophage in a concentration > 10 8 pfu/ml of the bacteriophage, (iii) evaluating the formation of phage plaques on the bacterial lawn; if no phage plaques were formed, the prokaryote is insensitive to the bacteriophage. Suitable procedures for a phage plaque assay for combinations of prokaryote and bacteriophage will be known to the person skilled in the art. In particular, a plaque assay may be performed using the protocol described by Mills et al. 2007“Efficient method for generation of bacteriophage insensitive mutants of Streptococcus thermophilus yoghurt and mozzarella strains”, which is hereby herein incorporated by reference.

Procedure 5: Plasmid curing. Plasmid curing techniques are known to the person skilled in the art, with many suitable reference materials. The person skilled in the art could, for example, consult Swarts et al 2012,“CRISPR Interference Directs Strand Specific Spacer Acquisition”, which is hereby herein incorporated by reference.

Procedure 6: Direct protein addition with cell-penetrating peptides. Direct protein addition with cell-penetrating peptides may be performed as described by Suresh et al , 2017,“Cell-Penetrating Peptide-Mediated Delivery of Cas9 Protein and Guide RNA for Genome Editing” in“Methods in Molecular biology”, which is hereby herein incorporated by reference.

Procedure 7: Sequence alignment. Amino acid sequence alignments can be obtained using BLASTp at the website of the National Center for Biotechnology Information. Two sequences may be aligned via BLASTp, especially using default algorithm parameters, such as using a BLOSUM62 matrix with a gap cost of 1 1 : 1 (existence:extension). Multiple sequence alignments may be obtained as described by Katoh and Standley 2013, “MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability, which is hereby herein incorporated by reference.

Procedure 8: BIM generation. The parent strain may be grown in liquid culture until an optical density at 600 mil of 0.2. The anti-CRISPR protein 115 may be provided to the parent strain (cells) via direct protein addition (optionally with cell- penetrating peptides) or via expression from a plasmid. Bacteriophage 125 may be added to the parent strain culture at a concentration equal to or higher than lO 1 pfu/ml and incubated at a suitable incubation temperature, such as 37 °C for S. thermophilus , especially with rocking. After 24h, bacterial cells may be recovered by centrifugation, washed twice with NaCl 0.9%, and resuspended in NaCl 0.9%. 10-fold serial dilutions of the cells may be made in NaCl 0.9% and plated in agar plates to obtain isolated colonies. The isolated colonies may be tested for phage sensitivity or resistance by growing them, making bacterial lawns, and adding a drop of bacteriophages at a concentration of 10 8 pfu/ml of the bacteriophage. The absence of plaque formation indicates resistance, and thus a BIM 250. The term ‘plurality refers to two or more. Furthermore, the terns“a plurality of’ and“a number of’ may be used interchangeably.

The terms“substantially” or“essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms“substantially” or“essentially” may also include embodiments with“entirely”,“completely”,“all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term“essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms“about” and“approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”,“about”, and“approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

The term “comprise” includes also embodiments wherein the term “comprises” means“consists of’.

The term“and/or” especially relates to one or more of the items mentioned before and after“and/or”. For instance, a phrase“item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term“comprising” may in an embodiment refer to “consisting of’ but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species".

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

In the claims, any reference signs placed between parentheses shall not be constaied as limiting the claim. Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words“comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”.

The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method respectively.

The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.