The present invention is related to the field of CRISPR-Cas9 gene editing platforms. In particular, the present invention has identified Type II-C Cas9 anti-CRISPR (Acr) inhibitors that control Cas9 gene editing activity. Co-administration of such Acr inhibitors may provide an advantageous adjunct in permitting safe and practical biological therapeutics through spatial or temporal control of Cas9 activity; controlling Cas9-based gene drives in wild populations to reduce the ecological consequences of such forced inheritance schemes; and contributing to general research into various biotechnological, agricultural, and medical applications of gene editing technologies. Background

CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats; CRISPR- associated system) comprises a bacterial immune system that recognizes and destroys foreign nucleic acids. The development of Type II CRISPR-Cas9 systems as programmable nucleases for genome engineering has been beneficial in the biomedical sciences. For example, a Cas9 platform has enabled gene editing in a large variety of biological systems, where both gene knockouts and tailor-made alterations are possible within complex genomes with unprecedented accuracy and efficiency. The CRISPR-Cas9 system has the potential for application to gene therapy approaches for disease treatment, whether for the creation of custom, genome-edited cell-based therapies or for direct correction or ablation of aberrant genomic loci within patients. Mutant versions of Cas9 in which the DNA cleavage activity has been inactivated [―dead‖ Cas9 (dCas9)] have also been developed for RNA-guided genome binding, enabling further applications in gene expression control and genome structure visualization.

The safe application of CRISPR-Cas9 in gene therapy requires an ability to control the gene editing activity of a Cas9/sgRNA complex once the intended use has been realized. While several engineered systems allow for controlled activation of CRISPR-Cas9 to increase precision, all of these systems still lack the ability to provide predictable control and robust inhibition. What is needed in the art is the ability to predictably control gene editing (Cas9) or genome binding (dCas9) activity to prevent unintended Cas9 cleavage or DNA binding activity once a specific goal has been attained. Additionally, the ability to restrict Cas9 cleavage activity to a particular site, tissue, or cell cycle stage would greatly improve the efficacy and safety of Cas9-based clinical treatments and research applications. Summary Of The Invention

The present invention is related to the field of CRISPR-Cas9 gene editing platforms. In particular, the present invention has identified Type II-C Cas9 anti-CRISPR (Acr) inhibitors that control Cas9 gene editing activity. Co-administration of such Acr inhibitors may provide an advantageous adjunct in permitting safe and practical biological therapeutics through spatial or temporal control of Cas9 activity; controlling Cas9-based gene drives in wild populations to reduce the ecological consequences of such forced inheritance schemes; and contributing to general research into various biotechnological, agricultural, and medical applications of gene editing technologies.

In one embodiment, the present invention contemplates a Type II-C anti-CRISPR (Acr) protein. In one embodiment, the Acr protein is a truncated protein. In one embodiment, the truncated protein is derived from an amino acid sequence including, but not limited to, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and/or SEQ ID NO: 16. In one embodiment, the Acr protein is a fusion protein. In one embodiment, the Acr fusion protein comprises a C-terminal adduct. In one embodiment, the Acr fusion protein comprises an N-terminal adduct. In one embodiment, the adduct is a nuclear localization sequence. In one embodiment, the adduct is an affinity tag. In one embodiment, the affinity tag is FLAG. In one embodiment, the Acr protein is a dimer protein. In one

embodiment, the Acr dimer protein is an Acr homodimer protein. In one embodiment, the Acr dimer protein is an Acr heterodimer protein. In one embodiment, the Acr protein is a mutated protein. In one embodiment, said Acr protein comprises at least a portion of an amino acid sequence selected from the group consisting of AcrIIC1Boe, AcrIIC1Nme, AcrIIC2Nme, AcrIIC3Nme, AcrIIC4Hpa, and AcrIIC5Smu. In one embodiment, said protein is less than approximately 14 kDa. In one embodiment, the present invention contemplates a composition comprising a Type II-C Cas9 protein comprising a binding site and a Type II-C anti-CRISPR (Acr) protein, wherein said Acr protein binds with specific affinity to said binding site. In one embodiment, the Type II-C Cas9 protein is a Brackiella oedipodis Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Neisseria meningitidis Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Haemophilus parainfluenzau Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Simonsiella muelleri Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Ralstonia solanacearum Cas9 protein. In one embodiment, the Acr protein is a truncated protein. In one embodiment, the truncated protein is derived from an amino acid sequence including, but not limited to, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and/or SEQ ID NO: 16. In one embodiment, the Acr protein is a fusion protein. In one embodiment, the Acr fusion protein comprises a C- terminal adduct. In one embodiment, the Acr fusion protein comprises an N-terminal adduct. In one embodiment, the adduct is a nuclear localization sequence. In one embodiment, the adduct is an affinity tag. In one embodiment, the affinity tag is FLAG. In one embodiment, the Acr protein is a dimer protein. In one embodiment, the Acr dimer protein is an Acr homodimer protein. In one embodiment, the Acr dimer protein is an Acr heterodimer protein. In one embodiment, the Acr protein is a mutated protein. In one embodiment, said type II-C Cas9 protein is a type II-C dCas9 protein. In one embodiment, said type II-C dCas9 protein is dNmeCas9.

In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a biological cell comprising at least one suspect gene; ii) a Type II-C Cas9/sgRNA complex wherein said sgRNA is capable of hybridizing to a target site of said at least one suspect gene and said Type II-C Cas9 protein comprises a binding site; and iii) a Type II-C anti-CRISPR (Acr) protein that binds with specific affinity to said binding site; b) contacting said Type II-C Cas9/sgRNA complex with said biological cell such that said Type II-C Cas9/sgRNA complex hybridizes to said target site; c) editing said suspect gene with said Type II-C Cas9/sgRNA complex; and d) contacting said binding site with said Acr protein such that said editing is reduced. In one embodiment, the biological cell is a bacterial cell. In one embodiment, the biological cell is a fungal cell. In one embodiment, the biological cell is a mammalian cell. In one embodiment, the biological cell is an insect cell. In one embodiment, the biological cell is a plant cell. In one embodiment, the reduced editing resulting from Acr protein action slows said gene drive propagation. In one embodiment, the suspect gene is a mutated gene. In one embodiment, the suspect gene is a dysregulated gene. In one embodiment, the suspect gene is a damaged gene. In one embodiment, the suspect gene is a dysfunctional gene. In one

embodiment, the Type II-C Cas9 protein is a Brackiella oedipodis Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Neisseria meningitidis Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Haemophilus parainfluenzau Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Simonsiella muelleri Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Ralstonia solanacearum Cas9 protein. In one embodiment, the target site is N-TS3. In one embodiment, the target site is N-TS7. In one embodiment, the target site is N-TS25. In one embodiment, the target site is D-TS3. In one embodiment, the target site is D-TS7. In one embodiment, the Acr protein is a truncated protein. In one embodiment, the truncated protein is derived from an amino acid sequence including, but not limited to, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and/or SEQ ID NO: 16. In one embodiment, the Acr protein is a fusion protein. In one embodiment, the Acr fusion protein comprises a C-terminal adduct. In one embodiment, the Acr fusion protein comprises an N-terminal adduct. In one embodiment, the adduct is a nuclear localization sequence. In one embodiment, the adduct is an affinity tag. In one embodiment, the affinity tag is FLAG. In one embodiment, the Acr protein is a dimer protein. In one embodiment, the Acr dimer protein is an Acr homodimer protein. In one embodiment, the Acr dimer protein is an Acr heterodimer protein. In one embodiment, the Acr protein is a mutated protein. In one embodiment, said Acr protein comprises at least a portion of an amino acid sequence selected from the group consisting of AcrIIC1Boe, AcrIIC1Nme, AcrIIC2Nme, AcrIIC3Nme, AcrIIC4Hpa, and AcrIIC5Smu. In one embodiment, said Type II-C Cas9 is selected from the group consisting of a Brackiella oedipodis Cas9 protein, a Neisseria meningitidis Cas9 protein, a Haemophilus influenzae Cas9 protein, a Simonsiella muelleri Cas9 protein, and a Ralstonia solanacearum Cas9 protein. In one embodiment, said type II-C Cas9 protein is a type II-C dCas9 protein. In one embodiment, said type II-C dCas9 protein is dNmeCas9. In one embodiment, said reduced editing prevents at least one off-target event of Cas9 binding. In one embodiment, said at least one off-target event is selected from the group consisting of DNA cleavage and DNA mutation. In one embodiment, said reduced editing occurs during theG1 phase of the cell cycle. In one embodiment, said reduced editing prevents mosaic genotypes. In one embodiment, said reduced editing inhibits a gene drive. In one embodiment, said reduced editing is selected from the group consisting of at least 70%, 75%, 90% or greater and 100%. In one embodiment, said reduced editing is precisely controlled. In one embodiment, the suspect gene is a mutated gene. In one embodiment, the suspect gene is a dysregulated gene. In one embodiment, the suspect gene is a damaged gene. In one

embodiment, the suspect gene is a dysfunctional gene.

In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a virus comprising at least one suspect gene; ii) a Type II-C Cas9/sgRNA complex wherein said sgRNA is capable of hybridizing to a target site of said at least one suspect gene and said Type II-C Cas9 protein comprises a binding site; and iii) a Type II-C anti-CRISPR (Acr) protein that binds with specific affinity to said binding site; b) contacting said Type II-C Cas9/sgRNA complex with said virus such that said Type II-C Cas9/sgRNA complex hybridizes to said suspect gene; c) editing said suspect gene with said Type II-C Cas9/sgRNA complex; and d) contacting said binding site with said Acr protein such that said editing is reduced. In one embodiment, the suspect gene is a mutated gene. In one embodiment, the suspect gene is a dysregulated gene. In one embodiment, the suspect gene is a damaged gene. In one

embodiment, the suspect gene is a dysfunctional gene. In one embodiment, the Type II-C Cas9 protein is a Brackiella oedipodis Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Neisseria meningitidis Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Haemophilus parainfluenzau Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Simonsiella muelleri Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Ralstonia solanacearum Cas9 protein. In one embodiment, the target site is N-TS3. In one embodiment, the target site is N-TS7. In one embodiment, the target site is N-TS25. In one embodiment, the target site is D-TS3. In one embodiment, the target site is D-TS7. In one embodiment, the Acr protein is a truncated protein. In one embodiment, the truncated protein is derived from an amino acid sequence including, but not limited to, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and/or SEQ ID NO: 16. In one embodiment, the Acr protein is a fusion protein. In one embodiment, the Acr fusion protein comprises a C-terminal adduct. In one embodiment, the Acr fusion protein comprises an N-terminal adduct. In one embodiment, the adduct is a nuclear localization sequence. In one embodiment, the adduct is an affinity tag. In one embodiment, the affinity tag is FLAG. In one embodiment, the Acr protein is a dimer protein. In one embodiment, the Acr dimer protein is an Acr homodimer protein. In one embodiment, the Acr dimer protein is an Acr heterodimer protein. In one embodiment, the Acr protein is a mutated protein. In one embodiment, said Acr protein comprises at least a portion of an amino acid sequence selected from the group consisting of AcrIIC1Boe, AcrIIC1Nme, AcrIIC2Nme, AcrIIC3Nme, AcrIIC4Hpa, and AcrIIC5Smu. In one embodiment, said Type II-C Cas9 is selected from the group consisting of a Brackiella oedipodis Cas9 protein, a Neisseria meningitidis Cas9 protein, a Haemophilus influenzae Cas9 protein, a Simonsiella muelleri Cas9 protein, and a Ralstonia solanacearum Cas9 protein. In one embodiment, said type II-C Cas9 protein is a type II-C dCas9 protein. In one embodiment, said type II-C dCas9 protein is dNmeCas9. In one embodiment, said reduced editing prevents at least one off-target event of Cas9 binding. In one embodiment, said at least one off-target event is selected from the group consisting of DNA cleavage and DNA mutation. In one embodiment, said reduced editingoccurs during the G1 phase of the cell cycle. In one embodiment, said reduced editing prevents mosaic genotypes. In one embodiment, said reduced editing inhibits a gene drive. In one embodiment, said reduced editing is selected from the group consisting of at least 70%, 75%, 90% or greater and 100%. In one embodiment, said reduced editing is precisely controlled. In one embodiment, the suspect gene is a mutated gene. In one embodiment, the suspect gene is a dysregulated gene. In one embodiment, the suspect gene is a damaged gene. In one embodiment, the suspect gene is a dysfunctional gene.

In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a mammal comprising at least one suspect gene; ii) a Type II-C Cas9/sgRNA complex wherein said sgRNA is capable of hybridizing to a target site of at least one suspect gene and said Type II-C Cas9 protein comprises a binding site; and iii) a Type II-C anti-CRISPR (Acr) protein that is capable of binding with specific affinity to said binding site; b)

administering said Type II-C Cas9/sgRNA complex to said mammal such that said Type II-C Cas9/sgRNA complex binds to said target site; c) editing said suspect gene with said Type II-C Cas9/sgRNA complex; and d) administering said Acr protein to said mammal such that said Acr protein binds with specific affinity to said binding site such that said editing is reduced. In one embodiment, the mammal is a human. In one embodiment, the mammal is a livestock species. In one embodiment, the mammal exhibits at least one symptom of a medical disorder or disease resulting from the presence of said suspect gene. In one embodiment, the editing of said suspect gene reduces said at least one symptom. In one embodiment, the suspect gene is a mutated gene. In one embodiment, the suspect gene is a dysregulated gene. In one embodiment, the suspect gene is a damaged gene. In one embodiment, the suspect gene is a dysfunctional gene. In one embodiment, the Type II-C Cas9 protein is a Brackiella oedipodis Cas9 protein In one embodiment, the Type II-C Cas9 protein is a Neisseria meningitidis Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Haemophilus parainfluenzau Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Simonsiella muelleri Cas9 protein. In one embodiment, the Type II-C Cas9 protein is a Ralstonia solanacearum Cas9 protein. In one embodiment, the target site is N-TS3. In one embodiment, the target site is N-TS7. In one embodiment, the target site is N-TS25. In one embodiment, the target site is D-TS3. In one embodiment, the target site is D-TS7. In one embodiment, the Acr protein is a truncated protein. In one embodiment, the truncated protein is derived from an amino acid sequence including, but not limited to, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and/or SEQ ID NO: 16. In one embodiment, the Acr protein is a fusion protein. In one embodiment, the Acr fusion protein comprises a C-terminal adduct. In one embodiment, the Acr fusion protein comprises an N-terminal adduct. In one embodiment, the adduct is a nuclear localization sequence. In one embodiment, the adduct is an affinity tag. In one embodiment, the affinity tag is FLAG. In one embodiment, the Acr protein is a dimer protein. In one embodiment, the Acr dimer protein is an Acr homodimer protein. In one embodiment, the Acr dimer protein is an Acr heterodimer protein. In one embodiment, the Acr protein is a mutated protein. In one embodiment, said Acr protein comprises at least a portion of an amino acid sequence selected from the group consisting of AcrIIC1Boe, AcrIIC1Nme, AcrIIC2Nme, AcrIIC3Nme, AcrIIC4Hpa, and AcrIIC5Smu. In one embodiment, said Type II-C Cas9 is selected from the group consisting of a Brackiella oedipodis Cas9 protein, a Neisseria meningitidis Cas9 protein, a Haemophilus influenzae Cas9 protein, a Simonsiella muelleri Cas9 protein, and a Ralstonia solanacearum Cas9 protein. In one embodiment, said type II-C Cas9 protein is a type II-C dCas9 protein. In one embodiment, said type II-C dCas9 protein is dNmeCas9. In one embodiment, said reduced editing prevents at least one off-target event of Cas9 binding. In one embodiment, said at least one off-target event is selected from the group consisting of DNA cleavage and DNA mutation. In one embodiment, said at least one off-target event occurs during a G1 phase cell cycle. In one embodiment, said reduced editing prevents mosaic genotypes. In one embodiment, said reduced editing inhibits a gene drive. In one embodiment, said reduced editing is selected from the group consisting of at least 70%, 75%, 90% or greater and 100%. In one embodiment, said reduced editing is precisely controlled. I In one embodiment, the suspect gene is a mutated gene. In one embodiment, the suspect gene is a dysregulated gene. In one embodiment, the suspect gene is a damaged gene. In one

embodiment, the suspect gene is a dysfunctional gene.In one embodiment, the present invention contemplates a kit, comprising: a) a first container comprising at least one Type II-C anti- CRISPR (Acr) protein; b) a second container comprising a Type II-C Cas9 protein; and c) a third container comprising an sgRNA. In one embodiment, said Acr protein comprises at least a portion of an amino acid sequence selected from the group consisting of AcrIIC1Boe,

AcrIIC1Nme, AcrIIC2Nme, AcrIIC3Nme, AcrIIC4Hpa, and AcrIIC5Smu. In one embodiment, said Type II-C Cas9 is selected from the group consisting of a Brackiella oedipodis Cas9 protein, a Neisseria meningitidis Cas9 protein, a Haemophilus influenzae Cas9 protein, a Simonsiella muelleri Cas9 protein, and a Ralstonia solanacearum Cas9 protein. In one embodiment, said type II-C Cas9 protein is a type II-C dCas9 protein. In one embodiment, said type II-C dCas9 protein is dNmeCas9. Definitions

To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as―a‖,―an‖ and―the‖ are not intended to refer to only a singular entity but also plural entities and also includes the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

One of skill in the art will understand the interchangability of terms designating the various anti-CRISPR proteins due to a lack of consistency in the literature and an ongoing effort in the art to unify such terminology. For example, as used herein the designation of Acr1-Bo is interchangable with AcrIIC1Boe and the designation of Acr2-Nm is interchangeable with AcrIIC2Nme. Also, as used herein, the designation of Acr88a-32 is interchangable with AcrE2. This type of equivalency may also be seen in the various other designations as used throughout the present application.

The term "about" as used herein, in the context of any of any assay measurements refers to +/- 5% of a given measurement.

As used herein, the term―intein‖ refers to a segment of a protein that is able to excise itself and join the remaining portions of the protein (e.g., the exteins) with a peptide bond in a process termed protein splicing. Inteins have also been called "protein introns". This process involves intein-mediated protein splicing that occurs after an intein-containing mRNA has been translated into a protein. Consequently, a precursor protein contains three segments— an N- extein followed by the intein followed by a C-extein. After splicing has taken place, the resulting protein contains the N-extein linked to the C-extein; this splicing product is also termed an extein.

The term―suspect gene‖ as used herein, refers to a gene of interest wherein editing of the gene may have beneficial consequences to the health of the cell in which the gene resides, or the health of the organism in which the cell resides. A suspect gene refers also to a gene of interest in a cell or organism wherein editing of the gene may provide biological insight in laboratory experiments. In general, a suspect gene may include but is not limited to a mutated gene, a dysregulated gene, a damaged gene, and/or a dysfunctional gene.

As used herein, the term―CRISPRs‖ or―Clustered Regularly Interspaced Short

Palindromic Repeats‖ refers to an acronym for DNA loci that contain multiple, short, direct repetitions of base sequences. Each repetition contains a series of bases followed by the same series in reverse and then by approximately 30 base pairs known as "spacer DNA". The spacers are short segments of DNA that are often derived from a bacterial virus or other foreign genetic element and may serve as a 'memory' of past exposures to facilitate an adaptive defense against future invasions. Doudna et al. ,―Genome editing. The new frontier of genome engineering with CRISPR-Cas9‖ Science.2014346(6213):1258096.

As used herein, the term―Cas‖ or―CRISPR-associated (cas)‖ refers to genes or derived proteins often associated with CRISPR repeat-spacer arrays. Doudna et al. ,―Genome editing. The new frontier of genome engineering with CRISPR-Cas9‖ Science.2014346(6213):1258096.

As used herein, the term―Cas9‖ refers to a nuclease from Type II CRISPR systems, an enzyme specialized for generating double-strand breaks in DNA, with two active cutting sites (the HNH and RuvC domains), one for each strand of the double helix. Jinek et al. combined tracrRNA and spacer RNA into a "single-guide RNA" (sgRNA) molecule that, mixed with Cas9, could find and cleave DNA targets through Watson-Crick pairing between the guide sequence within the sgRNA and the target DNA sequence. Jinek et al.,―A programmable dual-RNA- guided DNA endonuclease in adaptive bacterial immunity‖ Science.2012337(6096):816-821 Epub 2012 Jun 28.

As used herein, the term―catalytically active Cas9‖ refers to an unmodified Cas9 nuclease comprising full nuclease activity.

The term―binding site‖ as used herein refers to a Cas9 amino acid sequence having specific affinity for an Acr1 protein.

The term―target site‖ as used herein refers to a nucleic acid sequence flanking a suspect gene that hybridizes to an sgRNA sequence.

The term―nickase‖ as used herein, refers to a nuclease that cleaves only a single DNA strand, either due to its natural function or because it has been engineered to cleave only a single DNA strand. Cas9 nickase variants that have either the RuvC or the HNH domain mutated provide control over which DNA strand is cleaved and which remains intact. Cong et al., ―Multiplex genome engineering using CRISPR/Cas systems‖ Science.2013339(6121):819-823. Epub 2013 Jan 3.

The term―protospacer adjacent motif‖ (or PAM) as used herein, refers to a DNA sequence present on the target DNA molecule adjacent to the sequence matching the guide RNA spacer. The PAM is required for a Cas9/sgRNA to form an R-loop to interrogate a specific DNA sequence through Watson-Crick pairing of its guide RNA with the genome. The PAM specificity may be a function of the DNA-binding specificity of the Cas9 protein (e.g., a ―protospacer adjacent motif recognition domain‖ at the C-terminus of Cas9).

As used herein, the term―sgRNA‖ refers to single guide RNA used in conjunction with CRISPR associated systems (Cas). sgRNAs are a fusion of crRNA and tracrRNA and contain nucleotides of sequence complementary to the desired target site. Watson-Crick pairing of the sgRNA with the target site permits R-loop formation, which in conjunction with a functional PAM permits DNA cleavage or in the case of nuclease-deficient Cas9 allows tight binding to the DNA at that locus. The term―dimerization domain‖ as used herein, refers to a domain, either protein, polynucleotide that allows the associate of two different molecules. A dimerization domain can allow homotypic and/or heterotypic interactions. Dimerization domains can also be drug- dependent (i.e. depending on the presence of a small molecule in order to function). Ho et al., ―Dimeric ligands define a role for transcriptional activation domains in reinitiation‖ Nature.1996 382(6594):822-826.

As used herein, the term―edit‖―editing‖ or―edited‖ refers to a method of altering a nucleic acid sequence of a polynucleotide (e.g., for example, a wild type naturally occurring nucleic acid sequence or a mutated naturally occurring sequence) by selective deletion of a specific genomic target or the specific inclusion of new sequence through the use of an exogenously supplied DNA template. Such a specific genomic target includes, but may be not limited to, a chromosomal region, mitochondrial DNA, a gene, a promoter, an open reading frame or any nucleic acid sequence.

The term―delete‖,―deleted‖,―deleting‖ or―deletion‖ as used herein, may be defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are, or become, absent.

The term―suspected of having‖, as used herein, refers a medical condition or set of medical conditions (e.g., preliminary symptoms) exhibited by a patient that is insufficent to provide a differential diagnosis. Nonetheless, the exhibited condition(s) would justify further testing (e.g., autoantibody testing) to obtain further information on which to base a diagnosis.

The term―at risk for‖ as used herein, refers to a medical condition or set of medical conditions exhibited by a patient which may predispose the patient to a particular disease or affliction. For example, these conditions may result from influences that include, but are not limited to, behavioral, emotional, chemical, biochemical, or environmental influences.

The term―effective amount‖ as used herein, refers to a particular amount of a

pharmaceutical composition comprising a therapeutic agent that achieves a clinically beneficial result (i.e., for example, a reduction of symptoms). Toxicity and therapeutic efficacy of such compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD ₅₀ /ED ₅₀ . Compounds that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and additional animal studies can be used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The term―symptom‖, as used herein, refers to any subjective or objective evidence of disease or physical disturbance observed by the patient. For example, subjective evidence is usually based upon patient self-reporting and may include, but is not limited to, pain, headache, visual disturbances, nausea and/or vomiting. Alternatively, objective evidence is usually a result of medical testing including, but not limited to, body temperature, complete blood count, lipid panels, thyroid panels, blood pressure, heart rate, electrocardiogram, tissue and/or body imaging scans.

The term―disease‖ or―medical condition‖, as used herein, refers to any impairment of the normal state of the living animal or plant body or one of its parts that interrupts or modifies the performance of the vital functions. Typically manifested by distinguishing signs and symptoms, it is usually a response to: i) environmental factors (as malnutrition, industrial hazards, or climate); ii) specific infective agents (as worms, bacteria, or viruses); iii) inherent defects of the organism (as genetic anomalies); and/or iv) combinations of these factors.

The terms "reduce," "inhibit," "diminish," "suppress," "decrease,"―prevent‖ and grammatical equivalents (including―lower,‖―smaller,‖ etc.) when in reference to the expression of any symptom in an untreated subject relative to a treated subject, mean that the quantity and/or magnitude of the symptoms in the treated subject is lower than in the untreated subject by any amount that is recognized as clinically relevant by any medically trained personnel. In one embodiment, the quantity and/or magnitude of the symptoms in the treated subject is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and/or at least 90% lower than the quantity and/or magnitude of the symptoms in the untreated subject.

The term―inhibitory compound‖ as used herein, refers to any compound capable of interacting with (i.e., for example, attaching, binding etc) to a binding partner under conditions such that the binding partner becomes unresponsive to its natural ligands. Inhibitory compounds may include, but are not limited to, small organic molecules, antibodies, and proteins/peptides.

The term "administered" or "administering", as used herein, refers to any method of providing a composition to a patient such that the composition has its intended effect on the patient. An exemplary method of administering is by a direct mechanism such as, local tissue administration (i.e., for example, extravascular placement), oral ingestion, transdermal patch, topical, inhalation, suppository etc.

The term "patient" or―subject‖, as used herein, is a human or animal and need not be hospitalized. For example, out-patients, persons in nursing homes are "patients." A patient may comprise any age of a human or non-human animal and therefore includes both adult and juveniles (i.e., children). It is not intended that the term "patient" connote a need for medical treatment, therefore, a patient may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies.

The term―affinity‖ as used herein, refers to any attractive force between substances or particles that causes them to enter into and remain in chemical combination. For example, an inhibitor compound that has a high affinity for a receptor will provide greater efficacy in preventing the receptor from interacting with its natural ligands, than an inhibitor with a low affinity.

The terms "specific affinity" or―specifically bound" when used in reference to the interaction of a protein or peptide means that the interaction is dependent upon the presence of particular structures (i.e., for example, an target site, or binding site) on a protein determined by the teritiary and/or quaternary folding of a primary amino acid sequence. In other words, a protein or peptide is recognizing and binding to a particular conformational protein structure rather than to proteins in general.

The term―small organic molecule‖ as used herein, refers to any molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size from approximately 10 Da up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. The term―derived from‖ as used herein, refers to the source of a compound or sequence. In one respect, a compound or sequence may be derived from an organism or particular species. In another respect, a compound or sequence may be derived from a larger complex or sequence.

The term―test compound‖ as used herein, refers to any compound or molecule (including proteins) considered a candidate as an inhibitory compound.

The term―protein‖ as used herein, refers to any of numerous naturally occurring extremely complex substances (as an enzyme or antibody) that consist of amino acid residues joined by peptide bonds and contain the elements carbon, hydrogen, nitrogen, oxygen, usually sulfur. In general, a protein comprises amino acids having an order of magnitude within the hundreds.

The term―peptide‖ as used herein, refers to any of various amides that are derived from two or more amino acids by combination of the amino group of one acid with the carboxyl group of another and are usually obtained by partial hydrolysis of proteins. In general, a peptide comprises amino acids having an order of magnitude with the tens.

The term "polypeptide", refers to any of various amides that are derived from two or more amino acids by combination of the amino group of one acid with the carboxyl group of another and are usually obtained by partial hydrolysis of proteins. In general, a peptide comprises amino acids having an order of magnitude with the tens or larger.

The term "pharmaceutically" or "pharmacologically acceptable", as used herein, refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.

The term, "pharmaceutically acceptable carrier", as used herein, includes any and all solvents, or a dispersion medium including, but not limited to, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils, coatings, isotonic and absorption delaying agents, liposome, commercially available cleansers, and the like. Supplementary bioactive ingredients also can be incorporated into such carriers.

"Nucleic acid sequence" and "nucleotide sequence" as used herein refer to an

oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand. The term "an isolated nucleic acid‖, as used herein, refers to any nucleic acid molecule that has been removed from its natural state (e.g., removed from a cell and is, in a preferred embodiment, free of other genomic nucleic acid).

The terms "amino acid sequence" and "polypeptide sequence" as used herein, are interchangeable and to refer to a sequence of amino acids.

As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

The term "portion" when used in reference to a nucleotide sequence refers to fragments of that nucleotide sequence. The fragments may range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue.

A "variant" of a protein is defined as an amino acid sequence which differs by one or more amino acids from a polypeptide sequence or any homolog of the polypeptide sequence. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological or immunological activity may be found using computer programs including, but not limited to, DNAStar ^® software.

A "variant" of a nucleotide sequence is defined as a novel nucleotide sequence which differs from a reference oligonucleotide by having deletions, insertions and substitutions. These may be detected using a variety of methods (e.g., sequencing, hybridization assays etc.).

A "deletion" is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.

An "insertion" or "addition" is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues. A "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.

The term "derivative" as used herein, refers to any chemical modification of a nucleic acid or an amino acid. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group. For example, a nucleic acid derivative would encode a polypeptide which retains essential biological characteristics.

The term "biologically active" refers to any molecule having structural, regulatory or biochemical functions. For example, biological activity may be determined, for example, by restoration of wild-type growth in cells lacking protein activity. Cells lacking protein activity may be produced by many methods (i.e., for example, point mutation and frame-shift mutation). Complementation is achieved by transfecting cells which lack protein activity with an expression vector which expresses the protein, a derivative thereof, or a portion thereof.

The terms "binding component", "molecule of interest", "agent of interest", "ligand" or "receptor" as used herein may be any of a large number of different molecules, biological cells or aggregates, and the terms are used interchangeably. Proteins, polypeptides, peptides, nucleic acids (nucleotides, oligonucleotides and polynucleotides), antibodies, ligands, saccharides, polysaccharides, microorganisms such as bacteria, fungi and viruses, receptors, antibiotics, test compounds (particularly those produced by combinatorial chemistry), plant and animal cells, organs or fractions of each and other biological entities may each be a binding component. The term "macromolecule" as used herein, refers to any molecule of interest having a high molecular weight. For example, some biopolymers having a high molecular weight would be comprised of greater than 100 amino acids, nucleotides or sugar molecules.

The term "bind",―binding‖ or―bound‖ as used herein, includes any physical attachment or close association, which may be permanent or temporary. Generally, an interaction of hydrogen bonding, hydrophobic forces, van der Waals forces, covalent and ionic bonding etc., facilitates physical attachment between the molecule of interest and the analyte being measuring. The "binding" interaction may be brief as in the situation where binding causes a chemical reaction to occur. That is typical when the binding component is an enzyme and the analyte is a substrate for the enzyme. Reactions resulting from contact between the binding agent and the analyte are also within the definition of binding for the purposes of the present invention. Brief Description Of The Figures

Figure 1 presents exemplary data showing selective inhibition of NmeCas9 gene editing at the N-TS3 target site with several Acr proteins. NmeCas9+SgRNA plasmid (200 ng) + Acr plasmid (100 ng).

Figure 2 presents exemplary data showing selective inhibition of NmeCas9 gene editing at the N-TS25 target site with several Acr proteins. NmeCas9+SgRNA plasmid (200 ng) + Acr plasmid (200 ng).

Figure 3 presents exemplary data showing selective inhibition of NmeCas9 gene editing at the N-TS7 target site with several Acr proteins. NmeCas9+SgRNA plasmid (200 ng) + Acr plasmid (200 ng).

Figure 4 presents exemplary data showing selective inhibition of NmeCas9 (Type II-C) gene editing versus SpyCas9 (Type II-A) gene editing at the D-TS7 dual target site with several Acr proteins (both tagged and untagged). Nme/Spy Cas9+SgRNA plasmid (150 ng) + Acr plasmid (100 ng).

Figure 5 presents exemplary data showing selective inhibition of NmeCas9 (Type II-C) gene editing versus SpyCas9 (Type II-A) gene editing at the D-TS3 dual target site with several Acr proteins (some untagged). Nme/Spy Cas9+SgRNA plasmid (150 ng) + Acr plasmid (100 ng).

Figure 6 presents exemplary data showing a dose response relationship of Acr1Bo inhibition of NmeCas9 (Type II-C) gene editing at the D-TS3 target site. NmeCas9+SgRNA plasmid (150 ng) + Acr plasmid (150 ng).

Figure 7 presents exemplary data showing a dose response relationship of Acr1Nm inhibition of NmeCas9 (Type II-C) gene editing at the D-TS3 target site. NmeCas9+SgRNA plasmid (150 ng) + Acr plasmid (150 ng).

Figure 8 presents exemplary data showing a dose response relationship of Acr2Nm inhibition of NmeCas9 (Type II-C) gene editing at the D-TS3 target site. NmeCas9+SgRNA plasmid (150 ng) + Acr plasmid (150 ng).

Figure 9 presents exemplary data showing selective inhibition of NmeCas9 (Type II-C) gene editing versus SpyCas9 (Type II-A) gene editing at the D-TS3 dual target site with several Acr proteins (complete untagged set). Nme/Spy Cas9+SgRNA plasmid (150 ng) + Acr plasmid (100 ng). Figure 10 presents exemplary data showing a dose response relationship of Acr3Nm inhibition of NmeCas9 (Type II-C) gene editing at the D-TS3 target site. NmeCas9+SgRNA plasmid (150 ng) + Acr plasmid (150 ng).

Figure 11 presents exemplary data showing a titration of tagged Acr1Bo DTS3 site editing with Acr88-32 as a negative control using 150ng Cas9/150ng SgRNA.

Figure 11A: Acr1Bo with a C-terminal FLAG tag.

Figure 11B: Acr1Bo with a N-terminal FLAG tag.

Figure 12 presents exemplary data showing a titration of tagged Acr1Nm DTS3 site editing with Acr88-32 as a negative control using 150ng Cas9/150ng SgRNA.

Figure 12A: Acr1Nm with a C-terminal FLAG tag.

Figure 12B: Acr1Nm with a N-terminal FLAG tag.

Figure 13 presents exemplary data showing a titration of tagged Acr2Nm DTS3 site editing with Acr88-32 as a negative control using 150ng Cas9/150ng SgRNA.

Figure 13A: Acr2Nm with a C-terminal FLAG tag.

Figure 13B: Acr2Nm with a N-terminal FLAG tag.

Figure 14 presents exemplary data showing a titration of tagged Acr3Nm DTS3 site editing with Acr88-32 as a negative control using 150ng Cas9/150ng SgRNA.

Figure 14A: Acr3Nm with a C-terminal FLAG tag.

Figure 14B: Acr3Nm with a N-terminal FLAG tag.

Figure 15 presents a quantitative summary of the titration data showing that Acr adduct proteins (e.g., FLAG tagged) have similar activities as compared to Acr proteins without an adduct.

Figure 15A: Acr1Bo

Figure 15B: Acr1Nm

Figure 15C: Acr2Nm

Figure 15D: Acr3Nm

Figure 16 presents exemplary data showing direct binding of Acr proteins with a Cas9 protein. In particular, a Coomassie-stained SDS-PAGE analysis shows binding interactions between NmeCas9 and Acr1Nm, Acr2Nm, Acr3Nm, and Acr1Bo (indicated with *), with possible preliminary evidence for interactions between NmeCas9 and Acr6rs and Acr7pa (indicated with *?). AcrF1, a type I-F anti-CRISPR, is used as a negative control. Figure 17 presents exemplary data showing direct binding of Acr proteins with a

NmeCas9 protein. Input (i.e. total protein) and elution fractions are shown. AcrF1 (labelled here as 30-35), a type I-F anti-CRISPR, is used as a negative control.

Figure 18 presents exemplary data showing the simultaneous, non-competitive, binding of Acr1Nm and Acr2Nm to a NmeCas9 protein.

Figure 19 presents illustrative embodiments regarding the identification and validation of type II-C anti-CRISPR proteins.

Figure 19A: Schematic representation of candidate type II-C acr and aca genes within putative MGEs in the genomes of strains of Brackiella oedipodis and Neisseria meningitidis. Orthologous genes are color-matched, with percent amino acid identities indicated. Gene arrows are not drawn to scale. Any known, relevant gene product functions are annotated as follows: Rep, plasmid replication protein; Reg, transcriptional regulator; Tra, conjugal transfer protein; Rec, recombinase; Tail, phage tail structural protein; Lysis, phage lysis cassette. Genes colored in grey have MGE-related functions or show clear evidence of horizontal transfer.

Figure 19B: Schematic representation of genotypes in N. meningitidis strains used to test candidate anti- CRISPR function. Diamonds, CRISPR repeats; numbered rectangles, CRISPR spacers; arrows, CRISPR transcription. ermC, integrated

erythromycin resistance cassette; acrX, integrated candidate anti-CRISPR cassette.

Individual genetic elements are not to scale.

Figure 19C: Candidate type II-C anti-CRISPR proteins inhibit CRISPR interference in N. meningitidis. Results of the transformation assay in N. meningitidis strain 8013, and isogenic derivatives with each indicated acr gene integrated at the nics locus (see B), are plotted. The CRISPR-targeted protospacer plasmid (navy) cannot transform wild-type and empty vector-containing cells due to an active CRISPR-Cas system, resulting in zero transformants. BDL = below detection limit of this assay.

Plasmid DNA that lacks a target protospacer sequence can transform all strains equally well (yellow). Experiments were repeated three times and error bars represent the standard error of the mean (s.e.m.) between three replicates. Cells were also plated on non-selective media and the total number of cfu/mL present was equivalent in each sample (data not shown). Figure 20 presents a schematic of putative AcrIIC1 orthologs that are widely dispersed in MGEs of different species. The schematic representation of AcrIIC1 orthologs is identified by PSI-BLAST and their genomic contexts. The species in which each ortholog is found and its predicted genomic region classification (i.e., prophage, integrated conjugative element) are indicated. Gene arrows are not drawn to scale. Grey arrows represent genes that have a clear connection to mobile DNA; either by function (i.e. integrase) or by evidence of horizontal transfer as determined by BLAST search. Known, relevant gene functions are indicated by labels: Rep, plasmid replication protein; Reg, transcriptional regulator; Tra, conjugal transfer protein; Par, plasmid partitioning protein; H-NS, histone-like nucleoid-structuring protein; HTH, helix-turn-helix DNA-binding protein; Transp, transposase; Lysis, phage lysis cassette; Nuc, nuclease; Met, methyltransferase; RM, restriction modification system.

Figure 21 presents exemplary data showing that anti-CRISPR proteins bind directly to NmeCas9:sgRNA.

Figure 21A: Purified, untagged anti-CRISPR proteins were mixed with purified, 6xHis tagged NmeCas9:sgRNA in vitro. The input and elution fractions (before and after nickel affinity purification) are shown on the right and left sides of the Coomassie-stained SDS-PAGE gel, respectively. Mobilities of marker proteins (in kDa) are denoted on the left. AcrE2 is an inhibitor of the type I-E CRISPR-Cas system, and is included in this assay as a negative control.

Figure 21B: Binding assays were carried out between the same anti-CRISPR proteins tested in (A) and Cas9 from Actinomyces naeslundii (AnaCas9). AnaCas9 is a distantly related type II-C Cas9 protein (~20% amino acid sequence identity with NmeCas9).

Figure 22 redisplays the exemplary data of Figure 21 as uncropped images of the Coomassie-stained SDS-PAGE analysis.

Figure 23 presents exemplary data showing that type II-C Anti-CRISPR proteins specifically block in vitro DNA cleavage by NmeCas9. Linear plasmid DNA bearing a protospacer adjacent to a PAM sequence was subjected to in vitro digestion by purified, recombinant, sgRNA-programmed NmeCas9 (upper panel) or SpyCas9 (lower panel). Where indicated at the top of each lane, Cas9 was pre-incubated with purified anti-CRISPR proteins as indicated with AcrE2 as a negative control. Molar equivalents of anti-CRISPR protein (relative to Cas9) are shown at the top of each lane, and mobilities of input and cleaved DNAs are denoted on the right.

Figure 24 presents exemplary data showing that type II-C anti-CRISPR proteins specifically block genome editing by NmeCas9 in human cells.

Figure 24A: Schematic representation of R-loop structures at a dual target site (DTS3) in the human genome that can be cleaved and edited by either SpyCas9 (top) or NmeCas9 (bottom). Guide sequences (purple), PAMs (boxed), and Cas9 cleavage sites (red line) are indicated.

Figure 24B: T7E1 assays of NmeCas9 or SpyCas9 editing efficiencies at DTS3 upon transient transfection of human HEK293T cells. Constructs encoding anti-CRISPR proteins were cotransfected as indicated at the top of each lane. Mobilities of T7E1- digested (edited) and - undigested (unedited) bands are indicated to the right, and editing efficiencies (―% lesion‖) are given at the bottom of each lane.

Figure 25 presents exemplary data showing that AcrIIC1Boe blocks NmeCas9-Mediated Genome Editing at Multiple Human Genome Sites. Related to Figure 24.

Figure 25A: T7E1 assays of NmeCas9 editing efficiencies at multiple sites, with canonical (N4GATT) or variant (N4GTTT, N4GTCT) PAMs, upon transient transfection of human HEK293T cells. Plasmid encoding AcrIIC1Boe proteins was co-transfected as indicated at the top of each lane. For the D-TS7 target site, SpyCas9 editing (with and without AcrIIC1Boe) was also tested. Editing efficiencies (―% lesion‖) are given at the bottom of each lane.

Figure 25B: For the D-TS3 site tested in Figure 24, and for each site tested in Figure 25A, the NmeCas9 sgRNA spacer sequences (5’ to 3’) and DNA target sites (non- complementary strand, 5’ to 3’) are listed.

Figure 26 presents exemplary data showing plasmid titration of anti-CRISPR proteins in human genome editing. Related to Figure 24.

Figure 26A: T7E1 assays of NmeCas9 editing efficiencies at DTS3 upon transient transfection of human HEK293T cells. Constructs encoding anti-CRISPR proteins were co-transfected as indicated at the top of each lane. The total amount of anti-CRISPR proteins was held constant at 100 ng per well, but the relative amount of a negative control anti-CRISPR (AcrE2) and test anti-CRISPR (AcrIIC1Boe) was varied. Editing efficiencies (―% lesion‖) are given at the bottom of each lane.

Figures 26B-E: As in (A), except that AcrIIC1Nme (B), AcrIIC2Nme (C), and AcrIIC3Nme (D and E) were used. In (D), because inhibition was nearly complete even at the lowest dose (10 ng) of AcrIIC3Nme plasmid, we repeated the titration with lower levels of plasmid in (E), revealing the dose-dependence of inhibition.

Figure 27 presents exemplary data showing that AcrIIC2Nme and AcrIIC3Nme prevents DNA binding by NmeCas9 in human cells.

Figure 27A: Schematic representation of plasmids used for expression of dNmeCas9-(sfGFP) ₃ , dSpyCas9-(mCherry) ₃ , their respective telomeric sgRNAs, and anti-CRISPR proteins. The plasmid encoding the anti-CRISPR protein is also marked with the blue fluorescent protein mTagBFP2.

Figures 27B-F: Fluorescence images of U2OS cells transiently transfected with plasmids depicted in Figure 27A. The specific version of each plasmid set (with or without sgRNAs, with or without anti-CRISPRs) is given to the right of each row. First column: differential interference contrast (DIC) and mTagBFP2 imaging, merged. Second column: dNmeCas9-(sfGFP)3. Third column: dSpyCas9-(mCherry)3. Fourth column: dNmeCas9-(sfGFP)3 and dSpyCas9-(mCherry)3, merged. Scale bars, 5 μm. Figure 28 presents an exemplary analysis showing that anti-CRISPR proteins may interact with most, if not all, CRISPR-Cas systems.

Figure 28A: Sequence similarity tree of the interference proteins of a

representative set of CRISPR-Cas systems (Makarova et al., 2015). Class 1 systems are multi-subunit effector complexes, whereas Class 2 systems employ single effector proteins. Type and subtype clades are labelled. Stars indicate the types of CRISPR-Cas systems for which anti-CRISPR proteins have been discovered.

Figure 28B: A maximum likelihood phylogenetic tree of representative Cas9 protein sequences. Each protein is classified based on the CRISPR locus in which it resides as type II-A (blue), type II-B (yellow), or type II-C (purple). Cas9 proteins belonging to any genus that has a type II-C anti- CRISPR ortholog are coloured in red. With the assumption that a given anti-CRISPR ortholog inhibits the CRISPR-Cas system in the species where it is found, this visualization provides an estimate of the breadth of activity encompassed by the anti-CRISPR families discovered here.

Figure 29 presents exemplary data showing selective inhibition of NmeCas9 (Type II-C) gene editing vs. SpyCas9 (Type II-A) gene editing at the D-TS3 (top panel) and D-TS7 (bottom panel) dual target sites with three Acr proteins (AcrIIC3Nme (positive control), AcrIIC4Hpa and AcrIIC5Smu). Nme/Spy Cas9+SgRNA plasmids (150 ng each) + Acr plasmid (100 ng).

Figure 30 presents exemplary data showing a dose-response relationship of AcrIIC4Hpa (top panel) and AcrIIC5Smu (bottom panel) inhibition of NmeCas9 (Type II-C) editing at the D- TS3 dual target site. NmeCas9+SgRNA plasmids (150 ng each) + Acr plasmid (100 ng).

Figure 31 (panels A-E) presents fluorescence images of U2OS cells transiently transfected with plasmids depicted in Figure 27A. The specific version of each plasmid set (with or without sgRNAs, with or without anti- CRISPRs) is given to the right of each row. First column: differential interference contrast (DIC) and mTagBFP2 imaging, merged. Second column: dNmeCas9-(sfGFP)3. Third column: dSpyCas9-(mCherry)3. Fourth column:

dNmeCas9-(sfGFP)3 and dSpyCas9-(mCherry)3, merged. Scale bars, 5 μm. Panel F:

Quantitation of dNmeCas9-(sfGFP)3 telomeric foci, as judged by co-localization with dSpyCas9-(mCherry)3 telomeric foci, in cells that express no anti-CRISPR, negative control anti-CRISPR (AcrE2), AcrIIC3Nme, AcrIIC4Hpa, or AcrIIC5Smu. Foci were scored blind.

Figure 32 presents exemplary data showing that some Acr proteins affect the ability of NmeCas9 to accumulate when co-expressed in human cells, either without (upper panel) or with (lower panel) co-expressed sgRNA. HEK293 cells were transfected with NmeCas9+SgRNA plasmids (100 ng each) + Acr plasmid (200 ng). In each panel, HA-tagged NmeCas9 was detected by probing a western blot of total protein (20 micrograms per lane) from transfected cells, using anti-HA antibodies. Detection of GAPDH protein with an anti-GAPDH antibody was used as a loading control, as indicated.

Figure 33 shows that AcrIIC1, AcrIIC2, and AcrIIC3 bind to NmeCas9 at distinct interaction surfaces. Untagged AcrIIC3 _Nme was co-expressed with 6His-tagged NmeCas9 in E. coli. The complex of the two proteins was purified using Ni-affinity chromatography. Following purification, this NmeCas9/AcrIIC3 _Nme complex was incubated with purified, untagged

AcrIIC2 _Nme , or both proteins. NmeCas9 and bound proteins were then purified using a second Ni-affinity column and the resulting complexes were analyzed by SDS-PAGE followed by Coomassie staining. All three anti-CRISPR proteins are able to bind NmeCas9

simultaneously, and the binding of one did not abrogate the binding of any other. This indicates that they could be used simultaneously to inhibit the activity of Cas9 and decrease the chances of anti-CRISPR resistant mutants arising.

Figure 34 presents DNA binding by NmeCas9 in the absence and presence of anti- CRISPR proteins, assayed using an electrophoretic mobility shift assay. Purified

NmeCas9+sgRNA is present in all lanes but the first one. The relevant anti-CRISPR protein added to the reaction is noted above each lane. NmeCas9+sgRNA and the anti-CRISPR proteins were premixed and incubated on ice for 5 minutes, ³² P-labelled DNA was added, and the reactions were incubated for 30 minutes at 37°C. AcrIIC1 _Boe and AcrIIC5 _Smu completely block Cas9 DNA-binding, while AcrIIC4 _Hpa partially inhibits binding at the concentration used.

AcrIIC2 _Nme does not interfere with DNA binding, and appears to bind to the NmeCas9/DNA complex. AcrE2, an anti-CRISPR protein for the type I-E CRISPR system, does not inhibit DNA binding by NmeCas9.

Figure 35 shows a direct interaction of AcrIIC4Hpa with NmeCas9. Untagged

AcrIIC4Hpa was co-expressed with 6His tagged NmeCas9 and the complex was purified and visualized by SDS-PAGE followed by Coomassie staining. Detailed Description Of The Invention

The present invention is related to the field of CRISPR-Cas9 gene editing platforms. In particular, the present invention has identified Type II-C Cas9 anti-CRISPR (Acr) inhibitors that control Cas9 gene editing activity. Co-administration of such Acr inhibitors may provide an advantageous adjunct in permitting safe and practical biological therapeutics through spatial or temporal control of Cas9 activity; controlling Cas9-based gene drives in wild populations to reduce the ecological consequences of such forced inheritance schemes; and contributing to general research into various biotechnological, agricultural, and medical applications of gene editing technologies. I. CRISPR Gene Editing Platform

CRISPR-Cas systems are adaptive immune systems in bacteria and archaea that help defend against attack by predatory viruses. These systems use small RNAs [CRISPR RNAs (crRNAs)] as guides to identify their nucleic acid targets, which are then cleaved and inactivated. In most instances, the target nucleic acid is DNA, not RNA.

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) RNA sequences and CRISPR-associated (Cas) genes form catalytic protein-RNA complexes that utilize the incorporated RNA to generate sequence-specific double strand breaks at a complementary DNA sequence. This nuclease platform has displayed remarkable robustness for targeted gene inactivation or tailor-made genome editing. Sander et al., CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32, 347–355 (2014); Mali et al. RNA- guided human genome engineering via Cas9. Science 339, 823–826 (2013); Ran et al. Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity. Cell 154, 1380–1389 (2013); Fu et al., Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nature Biotechnology 32, 279-284 (2014); and Wang et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

The CRISPR/Cas9 genome engineering system is revolutionizing biological sciences due to its simplicity and efficacy ^1-3 . The most commonly studied Cas9 nuclease originates from Streptococcus pyogenes (SpyCas9) ⁴ . SpyCas9 and its associated guide RNA license a DNA sequence for cleavage based on at least two stages of sequence interrogation ^4-8 : i) compatibility of a PAM element with the specificity of the PAM-interacting domain, and ii) complementarity of a guide RNA sequence with the target site. Because it is straightforward to program Cas9 to cleave a desired target site through incorporation of a complementary single guide RNA

(sgRNA) ⁴ , a primary constraint on Cas9 targeting is the presence of a compatible PAM element ^4,9,10 . For example, a PAM-interacting domain of wild-type SpyCas9 (SpyCas9 ^WT ) preferentially recognizes a NGG element ⁴ , although it can inefficiently utilize other PAM sequences (e.g. nAG, nGA) ^9,11 . The simplicity of a SpyCas9/sgRNA system allows facile editing of genomes in a variety of organisms and cell lines ^1-3 . Target specificity may be a function of recognition by both the guide RNA (through Watson-Crick base pairing) and an inherent specificity of Cas9 through recognition of a neighboring motif (e.g., for example, a protospacer adjacent motif (PAM)).

A subset of CRISPR-Cas systems (those known as―Type II‖) employ a protein called Cas9, and this protein (along with its RNA guides) has been adapted as a revolutionary genome editing platform. Variants carrying mutations in its nuclease active sites have also been developed as RNA-guided DNA binding platforms to enable gene labeling and transcriptional control. Cas9 is already in wide use as a research tool, and it is also being used in a broad range of biotechnology applications, and in livestock, agriculture, and elsewhere.

In addition, CRISPR-Cas9 platforms are being developed as a potentially revolutionary therapeutic approach to many diseases, given its potential ability to modify genomic loci that contribute to disease, to destroy the genomes of pathogens, or to introduce therapeutically useful sequences (e.g., chimaeric antigen receptors for cancer immunotherapies). Numerous Cas9s from various sources have been developed and adapted for these purposes, including, but not limited to, Cas9s encoded by the genomes of certain strains of Streptococcus pyogenes, Streptococcus thermophilus, and Staphylococcus aureus. These three are all from a sub-type of CRISPR-Cas systems known as Type II-A.

Most reported applications thus far have used S. pyogenes Cas9 (SpyCas9), which is the best characterized version, and more recently, S. aureus Cas9 (SauCas9). In addition to these Type II-A Cas9s, genome editing applications have been developed using N. meningitidis Cas9 (NmeCas9; Type II-C). Hou et al.,―Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis‖ Proc Natl. Acad Sci. USA 110:15644-15649 (2013); and Zhang et al.,―DNase H activity of Neisseria meningitidis Cas9‖ Molecular Cell 60:242-255 (2015).

A. Cas9 Activity Modulation

One current limitation of Cas9 application is that its activity can be very difficult to control– it is generally active as long as it is present and has access to its crRNA guide usually in the form of a so-called single-guide RNA (sgRNA). This lack of control can introduce difficulties for many reasons. Most notably, although Cas9 is often highly accurate at editing only the genomic locus to which it is directed, off-target effects are not uncommon. Off-target Cas9 activity has an unintended consequence of introducing mutations to unintended sites. This is a concern for many purposes especially, but not exclusively, to clinical therapeutics. It is well established that the more functional Cas9/sgRNA that is present in a cell, and the longer that the Cas9/sgRNA persists, the greater the likelihood of off-target effects.

Although it is not necessary to understand the mechanism of an invention, it is believed that once ―on-target‖ genome edits have occurred, persistent Cas9/sgRNA can no longer recognize and edit the intended locus, thereby completing its intended purpose. Nonetheless, the still- functional Cas9/sgRNA complex can continue to find and edit off-target sites elsewhere.

In one embodiment, the present invention contemplates a composition that inhibits Cas9/sgRNA activity. Although it is not necessary to understand the mechanism of the invention, it is believed that these inhibitor compounds interact with active Cas9/sgRNA and function as an―off switch‖ that reduces Cas9/sgRNA activity to a minimal amount and/or for a duration necessary for its intended purpose. It is further believed that a Cas9/sgRNA inhibitor composition can limit Cas9/sgRNA activity to particular tissues or organs, or to any given set of user-defined conditions, by―tripping the off-switch‖ anywhere, at any time or under any circumstance when its activity is not desired.

The present embodiments have specific advantages over other potential ways to control Cas9/sgRNA activity and/or tissue specificity, such as: i) limiting Cas9/sgRNA production; ii) controlling Cas9/sgRNA dosage; iii) use of specific promoters or other gene expression control elements; iv) using transient or tissue-specific delivery modalities. The regulatory control imparted by these alternative approaches fail to be as precise and/or transient as necessary, and any Cas9/sgRNA that does get introduced remains active for as long as it is present.

Others have suggested various methods to control Cas9/sgRNA activity:

i) Cas9/sgRNA can be split into two separate half-proteins, with its dimerization into an active form dependent upon a small molecule (e.g. rapamycin). Zetsche et al.,―A split-Cas9 architecture for inducible genome editing and transcription modulation‖ Nature Biotechnol. 33:139-140 (2015). Here, the small molecule acts as an―on-switch,‖ and its withdrawal can substitute for an―off-switch‖. However, among other limitations, the effects of small molecule withdrawal are not immediate, depending on its off-rate from Cas9 and on the persistence of the small molecules that were previously delivered.

(ii) Cas9/sgRNA can be disrupted by a ligand-dependent intein, rendering Cas9 inactive until a ligand (e.g., 4-hydroxytamoxifen (4-HT)) is added to trigger intein autoexcision and Cas9 activation. Davis et al.,―Small molecule-triggered Cas9 protein with improved genome-editing specificity‖ Nature Chem. Biol.11:316-318 (2015). Again, this is an on-switch mechanism, and the withdrawal of 4-HT will prevent further production of active Cas9. However, there is still no off-switch, and previously produced Cas9 will remain present and active.

(iii) Cas9/sgRNA can be fused to exogenous protein domains that confer light-inducible localization to the nucleus, where the target genome resides. As long as light of the appropriate wavelength is present, the Cas9/sgRNA remains in the nucleus and gene editing continues. When the light is withdrawn, the Cas9/sgRNA translocates to the cytoplasm and nuclear gene editing stops. Nihongaki et al.,―Photoactivatable CRISPR-Cas9 for optogenetic genome editing‖ Nature Biotechnol.33:755-760 (2015). However, it is usually not possible to exert sufficient control over the presence or absence of the appropriate light especially in living plants and animals, or outside of a controlled environment such as a laboratory. Furthermore, a Cas9/sgRNA might still have access to the genome, for example, under conditions as when the nuclear envelope is absent or compromised, e.g. during mitosis.

Accordingly, the identification or development of effective Cas9 off-switches would be a useful improvement in the art. Because CRISPR-Cas systems defend against virus

(bacteriophage) predation, these systems impose a strong evolutionary pressure on viruses to develop mechanisms to evade or counteract CRISPR-Cas defensive functions.

B. Type I CRISPR-Cas Inhibitors

Type I CRISPR-Cas systems, in contrast to Cas9-based Type II systems, employ a large, multisubunit protein complex for DNA surveillance and cleavage. As such, Type I systems are not as promising for many gene editing platforms as they comprise a more complicated system. However, Type I systems are prevalent in bacteria and they provide immunity against the viruses that infect bacteria, known as bacteriophages. Bacteriophage genes have been identified that encode protein inhibitors (anti-CRISPR: Acr) of Type I CRISPR-Cas systems. Bondy-Denomy et al.,―Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system‖ Nature 493:429-432 (2013): Bondy-Denomy et al.,―Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins‖ Nature 526:136-139 (2015); and Pawluk et al.,―A new group of phage anti-CRISPR genes inhibits the Type I-E CRISPR-Cas system of Pseudomonas aeruginosa‖ mBio 5:e00896-14 (2014). Each Type I anti-CRISPR identified to date possesses specificity to either the Type I-E or Type I-F subtypes of CRISPR-Cas systems, and therefore would not be expected to have an effect on any Cas9-based Type II systems. The Type I Acr proteins that have been characterized reportedly function by a direct, inhibitory binding to components of the CRISPR-Cas machinery, and each distinct Acr protein reportedly functions by a distinct mechanism of action. II. Type II Cas9 Inhibitor Compositions

In addition to phage-encoded Acr proteins that prevent Type I CRISPR-based immunity, the identification of Acr-associated (Aca) transcriptional regulators of the helix-turn-helix xenobiotic response element (HTH-XRE) family have also come to light. Aca transcriptional regulators are believed to regulate Acr protein expression, and are commonly encoded within phage/prophage genomes by adjacent, small open reading frames. By using known Aca protein sequences as bioinformatic search queries, additional Aca proteins have been identified. Many of these newly identified Aca genes are encoded by bacteriophages infecting species with Type I CRISPR-Cas systems. Small, previously uncharacterized open reading frames found adjacent to aca genes were shown to possess anti-CRISPR activity against Type I-F and I-E systems.

In one embodiment, the present invention contemplates a composition comprising an aca gene within a Brackiella oedipodis genome. In one embodiment, the Brackiella oedipodis genome comprises a Type II-C CRISPR-Cas locus. Although it is not necessary to understand the mechanism of an invention, it is believed that the Brackiella oedipodis Type II-C CRISPR- Cas locus is the category that includes NmeCas9, and is not analogous to a Type I CRISPR-Cas locus. In fact, it has been determined that the Brackiella oedipodis Cas9 locus is ~47% identical to NmeCas9.

Furthermore, it has been found that the B. oedipodis Aca protein is adjacent to an uncharacterized ORF that encodes a candidate Acr protein (Acr1Bo). In one embodiment, the present invention contemplates a plurality of Type-II Aca genes located within a plurality of phages, each of which is adjacent to small uncharacterized ORF that encodes a candidate Acr protein, for example: 1. Brackiella oedipodis Acr (Acr1Bo)

Open Reading Frame (276 nucleotides):

ATGaaagaggtatttaaattaaaaccagagctagtgacttataaaggttgcggctgg gcactggc

gtgcatcaaagacggagagatcatcgatctcacctacgttcgtgaccttggtattga agaatatg atgaaaacttcgatggccttgaacctgaaatcatctattacgatgttgtggcttctcaag cgtgc

aaagaagtcgcctatcgttatgaagaaatgggcgaatttaccttcggcttatgctcg tgttggga

attcaatgtaatgTAG (SEQ ID NO: 1)

Protein (91 amino acids):

MKEVFKLKPELVTYKGCGWALACIKDGEIIDLTYVRDLGIEEYDENFDGLEPEIIYY DVV ASQACKEVAYRYEEMGEFTFGLCSCWEFNVM (SEQ ID NO: 2)

2. Neisseria meningitidis Acr #1 (Acr1Nm)

Open Reading Frame (258 nucleotides):

ATGaataaaacttataaaattggaaaaaatgccgggtatgatggctgcggtctttgt cttgcggc

catttctgaaaatgaagctatcaaagttaagtatttgcgcgacatttgtcctgatta cgatggcg

atgataaagctgaggattggctgagatggggaacggacagccgcgtcaaagcagccg ctcttgaa

atggagcagtacgcatatacgtcggttggtatggcctcatgttgggagtttgttgaa ctaTGA (SEQ ID NO: 3)

Protein (85 amino acids):

MNKTYKIGKNAGYDGCGLCLAAISENEAIKVKYLRDICPDYDGDDKAEDWLRWGTDS RVKAAALEMEQYAYTSVGMASCWEFVEL (SEQ ID NO: 4)

3. Neisseria meningitidis Acr #2 (Acr2Nm)

Open Reading Frame (372 nucleotides):

ATGagcaaaaacaatattttcaacaagtatccaacaattattcacggcgaagcgcgg ggggagaa

tgacgaatttgtggtgcatacgcgctacccgcgattcttggcgcggaaatcttttga cgacaatt

tcacgggcgaaatgcctgcaaaacctgttaacggggaattgggacaaatcggcgaac cgcgccgc

cttgcttatgattcacggcttggtttgtggctttctgacttcatcatgttggacaac aacaagcc

gaaaaacatggaggattggcttgggcaattaaaagccgcctgcgatcgaatcgcggc ggatgatt

tgatgctgaatgaagatgcggcggatttggagggctgggatgatTGA (SEQ ID NO: 5)

Protein (123 amino acids):

MSKNNIFNKYPTIIHGEARGENDEFVVHTRYPRFLARKSFDDNFTGEMPAKPVNGEL GQI GEPRRLAYDSRLGLWLSDFIMLDNNKPKNMEDWLGQLKAACDRIAADDLMLNEDAAD LEGWDD (SEQ ID NO: 6) 4. Neisseria meningitidis Acr #3 (Acr3Nm)

Open Reading Frame (351 nucleotides):

ATGttcaaacgcgctattatcttcacttctttcaacggctttgaaaaagtttctcga actgaaaa acgccgccttgccaaaatcatcaatgctcgagtttccatcatcgacgaatacttgagagc caaag

acaccaacgcatcgcttgacggtcagtaccgcgctttcttgttcaacgacgaatcgc ccgcaatg

accgaatttctggcaaaacttaaagcctttgccgaaagttgcaccggaatcagcatc gacgcatg

ggaaattgaagaaagcgaatacgtccgcctgccggtggaacgcagggatttcttagc ggcagcca

acggcaaagagatttttaaaattTAA (SEQ ID NO: 7)

Protein (116 amino acids):

MFKRAIIFTSFNGFEKVSRTEKRRLAKIINARVSIIDEYLRAKDTNASLDGQYRAFL FNDE SPAMTEFLAKLKAFAESCTGISIDAWEIEESEYVRLPVERRDFLAAANGKEIFKI (SEQ ID NO: 8)

5. Neisseria meningitidis Acr #4 (Acr4Nm)

Open Reading Frame (267 nucleotides)

ATGgcaaaaggtagaacaagcattacagagcggctcaaaaagagccaaaaacgagag gcgcgccg

tgatatggcgcacgaatgggcggaaaaatgggagcaggattatttgagcctgctctc tcaaatca

aacaggcaatcagcaaaggacacgatgacgagcttatcgacttatttgctgatttac gcgcgctg

caacagccaaaatttgaggcattgcatcgagtgattgatgagcttatcacgccgaca cgggagct

tataTGA (SEQ ID NO: 9)

Protein (88 amino acids)

MAKGRTSITERLKKSQKREARRDMAHEWAEKWEQDYLSLLSQIKQAISKGHDDELID LF ADLRAL QQPKFEALHRVIDELITPTRELI (SEQ ID NO: 10)

6. Neisseria meningitidis Acr #5 (Acr5Nm)

Open Reading Frame (192 nucleotides)

ATGaaatacgccaaatcaaaatctatcagcaaaatcggtcaatatcatcaaactttt aaaatcct

ttgggataaactaccaaaagaattgattgagaaatcaacagccaaaaatctcgccat tattattg

atttgatgtatgagcaaaaagaatatggccatacagaggcatggcgcgaattaacat caTAA (SEQ ID NO: 11)

Protein (63 amino acids)

MKYAKSKSISKIGQYHQTFKILWDKLPKELIEKSTAKNLAIIIDLMYEQKEYGHTEA WRE LTS (SEQ ID NO: 12)

7. Ralstonia solanacearum Acr (Acr6Rs)

Open Reading Frame (312 nucleotides)

ATGtacgcaatctacacggacgcaggaatcctggcagtagcccccactgtggaggca gcgatcga

acgggccaggtgcgagcatgggctaaaagccgctcttgtggctcagaacacgagctg catctacc acggcgggatcgttgatttgaccactgcatggcgaatctctgccgattcagagcgagccg gagat

tcggagacgaaattgcgccgctgctcggcgcgcttggcggcagcagtagaggctggg cgacttcc

cgtgttcgctgtgatggcgcatggcgagttagatctaatcgaggatgccTGA (SEQ ID NO: 13)

Protein (103 amino acids)

MYAIYTDAGILAVAPTVEAAIERARCEHGLKAALVAQNTSCIYHGGIVDLTTAWRIS AD SERAGDSETKLRRCSARLAAAVEAGRLPVFAVMAHGELDLIEDA (SEQ ID NO: 14) 8. Pseudomonas aeruginosa Acr (Acr7Pa)

ORF (261 nucleotides)

ATGctggagcgatgcctccagattgtcaccacgccgggcgctgtgccccgagatcag gcggaggc

caacgtttgccgtttggccgggatgatcgtggacggcaggtatccagtggcaggcaa acgactgt

ctgatgcggctgcgacctactttgccgaccatccagagcagcaagtgccgtccgccg aggtcgcc

cggcgcggctggatcataaacgccccacgcctccggacgcgactggagcgcctgctg ggcgggTAA (SEQ ID NO: 15) Protein (86 amino acids)

MLERCLQIVTTPGAVPRDQAEANVCRLAGMIVDGRYPVAGKRLSDAAATYFADHPEQ Q VPSAEVARRGWIINAPRLRTRLERLLGG (SEQ ID NO: 16) 9. Haemophilus parainfluenzae Acr (AcrIIC4 _Hpa ) ORF (267 nucleotides): ATGaagatcaccagcagcaacttcgcgaccattgcgaccagcgagaactttgcgaagctg agcgtgctgccgaaaaaccaccgtgag ccgatcaagggtctgttcaaaagcgcggttgaacagtttagcagcgcgcgtgacttcttt aagaacgagaactacagcaaagagctggcgg aaaagttcaacaaagaagcggtgaacgaggcggttgaaaagctgcaaaaagcgatcgatc tggcggaaaaacagggcattcaatttTG A (SEQ ID NO: 22) Protein (88 amino acids): MKITSSNFATIATSENFAKLSVLPKNHREPIKGLFKSAVEQFSSARDFFKNENYSKELAE K FNKEAVNEAVEKLQKAIDLAEKQGIQF (SEQ ID NO: 23) 10. Simonsiella muelleri Acr (AcrIIC5 _Smu ) ORF (393 nucleotides): ATGaacaacagcatcaagttccacgtgagctacgacggtaccgcgcgtgcgctgtttaac accaaggagcaggcggaaaaatactgcc tggttgaggaaattaacgatgagatgaacggctataagcgtaaaagctgggaggaaaagc tgcgtgaggaaaactgcgcgagcgtgcag gactgggttgagaagaactacaccagcagctatagcgacctgttcaacatctgcgagatt gaagtgagcagcgcgggtcaactggttaaga tcgacaacaccgaggtggacgatttcgttgaaaactgctatggctttaccctggaggacg atctggaggaattcaacaaggcgaaacagta cctgcaaaaattttatgcggagtgcgaaaacTGA (SEQ ID NO: 24) Protein (130 amino acids): MNNSIKFHVSYDGTARALFNTKEQAEKYCLVEEINDEMNGYKRKSWEEKLREENCASV QDWVEKNYTSSYSDLFNICEIEVSSAGQLVKIDNTEVDDFVENCYGFTLEDDLEEFNKA KQYLQKFYAECEN (SEQ ID NO: 25) In one embodiment, the present invention contemplates a composition comprising a Type II CRISPR-Cas9 inhibitor (e.g., an Acr). In one embodiment, the Type II Acr is Acr1Bo, that inhibits a Type II-C CRISPR-Cas system. In one embodiment, the Type II Acr is derived from an N. meningitidis strain. In one embodiment, the present invention contemplates a Type II Acr including, but not limited to, AcrIIC1Boe, AcrIIC1Nme, AcrIIC2Nme, AcrIIC3Nme,

AcrIIC4Hpa, and AcrIIC5Smu. The data presented herein demonstrates that the Type II Cas9 inhibitors AcrIIC1Boe, AcrIIC1Nme, AcrIIC2Nme, AcrIIC3Nme, AcrIIC4Hpa, and AcrIIC5Smu control N. meningitidis CRISPR-Cas system immune function.

Tests and readouts for NmeCas9 editing, including inactivation of fluorescent reporter genes, as well as standard T7E1 assays (e.g. in the attached figures) to detect the introduction of genomic lesions have already been established. For the latter, HEK293T cells were transiently transfected with plasmids encoding NmeCas9 (fused to epitope tags and nuclear localization sequences) and sgRNAs that target sites adjacent to a functional protospacer adjacent moiety (PAM), usually NNNNGATT, though some variations from that consensus are permissible. Zhang et al.,―DNase H activity of Neisseria meningitidis Cas9‖ Molecular Cell 60:242-255 (2015); and Hou et al.,―Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis‖ Proc Natl. Acad Sci. USA 110:15644-15649 (2013). Representative genomic target sites used in these preliminary experiments are as follows, showing the entire PCR amplicon: 24-nt NmeCas9 target spacer: Bold

20-nt Spy target spacer: Italic

Protospacer Adjacent Moiety: Underlined (NNNNGAT(A/T) for NmeCas9; NGG for SpyCas9) Target Site N-TS3

CATTTCTTTATATGTCTTAATGCTGACCTTCTGCACAAATGCACCACTATACCATTA C CAGTTATTACCAGCTAATAGGGTGGGAGCTAATGAACACTTACAACTCTGTCCTCAG GAAAGTGCAGAGAAATTCATGCATCCCAGGAGGGGATGCTCAGAAAGAGGAAGCT GGTTTATGATTGGACTGCGTGGGCGTTTGCAAAGCAAGGTTTCATTGAAAAGAGATG TTTTCTTGTGGGGCATTTGAGTCAATTACCAAAGTCTATTTTTAAAACTTCTCCATAT GAGCCTGATCTATCTCTGAAGTTGTTTTGAAGACCACAGGACTGCTTGTAACATGTG CCATTGCCATTCTGCTTTTTATTCTTTTGATTGGAAGGACTAAAATGATTTTCACTTA

(SEQ ID NO: 17)

Target Site N-TS7

GGACAGAAGAGAGTAGGGAGACGAGAAGGCGGAGGACAGAAGAAATGGGGGAGA GGGAAGAGGACAGAGAGGCTGCGCGCCTCTGAATACGCCAAGTCCAGCAGAGCTG GAGGCCTGTGAGAGGAGCTGCAAGCTTGAGCAAAGGGAGAGAGGTGAGCGGATGAA GGGAGATTGGTGAGTATCCGCCCACGCACCTACTTGTAAAAAGATCAAGGGGAAAC ACGCAGAAGGTCCCGCGGGAGTCCTGTGACCCACGTGAGGTGCTCGTGCCAGCGCG GGGTGGGAGGTGGTGGGCAATGTTCGTCGTGGAGTTGAGGAAGAAATTCTCCAGCC TTAAGGAAGCAAAAGAGTTCAAAGATCAGTGAGGCTGCTCAACAGAGGGATATGCA GATGACAGAATGC (SEQ ID NO: 18)

Target Site N-TS25

GCAATCCACCCAATGCTAACTGGGATGTTTGATTTTGCAGCCTCTTTCAGAGCAGTT GCTAAAAGTGGCTCCACATCATAAGAGGGCTTCCCTCCCTCAATCCATGAGAGCAAC TAGGTTTTGCATCAGTGAAAGGAAGAAAAAGCAGGAGTTTGGCAAGAGGCTGCAAA GAGACGGCACTGGGCTCCACTAGAGTTCCTTCCCACCGCGTTTCTCATCCTGTCTTCT GCCTAGTGGATATGTCTGCGTGGGCGTGCACACACATTGGCTGATGAAACCCCCTTC CTGTTGCACAGGGTCAGAACTAAGCGAGGTGGGTGTAGCTTTGGAGGGTTCTGAAA TCTAAGAACCAGCTTCCTTTCCCACCGCTTTCCGCTGAGTCAGTTCACTGCAGAGTG CTCTGCAGGATCTGGAGGCCTTTGTGTTCA (SEQ ID NO: 19)

Target Site D-TS3

GGACAAAAGCAGCCCATTAGGACCCCCCCACACTCGCACCTCTCCCTGCCAAGACCT CTAGGAGCAGCAGGGCGGAGAGACAGAGCCTGCCGGTTGGCATGGAACAAACTGA CTGAAGGCGAGGTCCGGGGCGGAGGGGATTGGGTTGTAGGGCTGTGGAGGAGGGGC GGCGGAGGGGGCGCTGGGGCTCTCGCTTGCTTCAGCCCAGCCCTTCTAGTCAGCCCG CGACAACTCGCGCCAGCTACGGGGCCTCAGAGAAGCCGGACTTCGCAAGCACCATG CAGTGGATAAGGGGCGGATCGGGAATGGTGAGTGCATGTAACCTTGGCTTCCCTTGC TTGAGCCTCTCAGTCCCCCAGCCCCACCTCCAGTTCCTCCAACGAGCCACAAGGCAG TGAGCACCCTGGCCTCTGCCCACCGCCCTAGCCGCCGTCCTTGAGACACCAGTGAGC TTGCTGTGGCCATTTTAGGAGTCC (SEQ ID NO: 20)

Target Site D-TS7

AGGACTGCTCTCAGCTACCGGCCTCCTCTGGATGACGGGACTGCAGGAACCACGAG AACCCCAGTTCTAGCTCCCGGGGTGGGCAGGCTGCTTGGCAGGCAGGCCGCCTTCCC TCCACCAGGAGTCAGGTCTCCAGCCAGAGGTCCTGACCCAGGGCACAAGTGCTCGC ACTGGGAAGCAGGCCTCTGAGGCAGGACGTCTTCTCCTGTGGTGGAGTGGGGGTGT GGGCAGGGCAGGGAGGCCAGCAGAGAGAGGCTCGGGGAGCAGGCTCTGTGGGCTT GCAGGAGGCAGGTCTGTGGCCCCTCCCTGGACCCTAGCCTAATGCCCCCTGCACCCC ATGCCTATGTTCCAGCTTCCTGGGTCTGCAGGTCCAGCCGGCTGGCACCCTCCATGTA CCCAGGGGAGATTCCAGCCAGACACCCGCCCCCCGGCCCTGGCTAAGAAGTTGCTTC CTGTTGCCAGCATGACCTACCCTCGCCTCTTTGATGCCATCCGCTGCCACCTCCTTTT GCTCCTGGACCCTTTAGCCTCTCTGCCCTT (SEQ ID NO: 21) Genomic DNA was extracted and a fragment containing the target site was subjected to polymerase chain reaction amplification. The resultant amplicons were heat-denatured and re- annealed to form heteroduplexes. The heteroduplexes were then digested with T7E1 that cleaves DNA containing a mismatch or bulge. The amount of T7E1 digestion reflects the degree of heteroduplex formation, which in turn reflects the efficiency of mutagenesis at the target site by NmeCas9. The generated plasmids each expressed an Acr for testing. As a negative control, plasmids were generated that expressed a previously characterized, similarly sized Acr (Acr88a- 32: 84 amino acids). Acr88a-32 specifically inhibits an unrelated Type I-E CRISPR-Cas system and would be expected to have no effect on any form of Cas9. Pawluk et al.,―A new group of phage anti-CRISPR genes inhibits the Type I-E CRISPR-Cas system of Pseudomonas aeruginosa‖ mBio 5:e00896-14 (2014).

Each individual Acr was expressed from a separate plasmid, each in one of three forms: i) the native phage-encoded sequence (with no additional amino acids appended); ii) with an N- terminal fusion of a nuclear localization signal (NLS) and a FLAG epitope tag; and iii) with a C- terminal fusion of an NLS and a FLAG tag. Although it is not necessary to understand the mechanism of the invention, Acrs of between 85 to 123 amino acids are believed to be well below the size limit for passive protein diffusion through the nuclear pores. The data presented herein shows that with an NmeCas9 target site N-TS315-20% editing efficiencies were observed during standard non-inhibited NmeCas9 editing assays. At one-half the relative amounts/ratios of transfected plasmids encoding Type II CRISPR-Cas inhibitor proteins, reasonably strong inhibitory effects were seen with untagged Acr1Bo and Acr1Nm, and mild effects are seen with untagged Acr2Nm. Partial inhibitory effects were observed with tagged Acrs, especially C-terminally-tagged Acr1Bo (―Acr1Bo/Flag-NLS‖). The negative control (Acr88a-32) has no consistent inhibitory effect. These results suggest that the putative Type II-C inhibitors limit NmeCas9 editing efficiency, but might need to be expressed in higher amounts (relative to NmeCas9) for full inhibition to be observed. The data also suggests that some of the tags might be compatible with Acr function. See, Figure 1.

Additional tests with different target sites (e.g, N-TS25 and N-TS7) were performed with equivalent amounts of Acr plasmids that revealed robust NmeCas9 inhibition. All three untagged Acr proteins were strongly inhibitory, as was a C-terminally-tagged Acr1Bo. Other tagged Acrs showed partial effects, whereas the negative control Acr88a-32 is non-functional. See Figures 2 and 3, respectively.

A―dual‖ target site was edited (e.g., D-TS7) where―dual‖ refers to the fact that the site can be targeted by either SpyCas9 (Type II-A) or NmeCas9 (Type II-C). The capacity for dual targeting results from the fact that dual site flanking sequences (e.g., for example, GGGAGATT) matches the NGG PAM consensus for SpyCas9, as well as the NNNNGATT PAM consensus for NmeCas9. Therefore, this similarity allows a direct comparison of the relative inhibitory effects of Acr proteins on SpyCas9 and NmeCas9 on the exact same site, under otherwise equivalent conditions. Also, SpyCas9 and NmeCas9 expression constructs were used that had identical promoters, terminal fusions, etc. to facilitate direct comparisons. The results show that various Type II Acr inhibitors demonstrated strong inhibition of NmeCas9 editing activity, but no inhibition of SpyCas9 activity at the same site. This shows that the Type II Acr inhibitors (both tagged and untagged) are likely specific for Type II-C systems and cannot function on the distantly related Type II-A system. See, Figure 4. A second dual target site, D-TS3, was tested using only untagged Acrs showing similar results. See, Figure 5.

Further tests using NmeCas9 editing of the D-TS3 target site with either untagged Acr1Bo, Acr1Nm, or Acr2Nm shows that the inhibitory effect of these Acrs on NmeCas9 editing is dose-dependent. See, Figures 6, 7 and 8, respectively. The inhibitory effects of both Acr1Bo and Acr1Nm were observed to be complete. However, the inhibitory effect of Acr2Nm was incomplete.

The dual target site, D-TS3 was tested with complete set of untagged Acrs, unlike the above data presented in Figure 5. The tests reveal Acr3Nm to be an effective NmeCas9 inhibitor, while having no effect on SpyCas9 inhibition. See Figure 9.

Figure 10 presents exemplary data showing a dose response relationship of Acr1Bo inhibition of NmeCas9 (Type II-C) gene editing at the D-TS3 target site. NmeCas9+SgRNA plasmid (150 ng) + Acr plasmid (150 ng).

A dose response relationship between Acr3Nm and NmeCas9 editing of the D-TS3 target site shows that complete inhibition occurs even with very small amounts of transfected Acr3Nm plasmid, suggesting that Acr3Nm may be a particularly potent inhibitor. See, Figure 10.

In conclusion, these data demonstrate that various NmeCas9 inhibitors can prevent NmeCas9 gene editing activity in human cells. Although it is not necessary to understand the mechanism of the invention, it is believed that these Acr inhibitors represent Cas9 genome editing―off-switches‖.

A. Truncated Acr Proteins

In one embodiment, the present invention contemplates a truncated nucleic acid sequence encoding an Acr protein derived from the group including, but not limited to, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15.

In one embodiment, the present invention contemplates an Acr protein comprising a truncated amino acid sequence derived from the group including, but not limited to, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16.

B. Acr Dimer Proteins

In one embodiment, the present invention contemplates a homodimer Acr protein selected from the group including, but not limited to, (SEQ ID NO: 2)-(SEQ ID NO: 2), (SEQ ID NO: 4)-(SEQ ID NO: 4), (SEQ ID NO: 6)-(SEQ ID NO: 6), (SEQ ID NO: 8)-(SEQ ID NO: 8), (SEQ ID NO: 10)-(SEQ ID NO: 10), (SEQ ID NO: 12)-(SEQ ID NO: 12), (SEQ ID NO: 14)- (SEQ ID NO: 14), and/or (SEQ ID NO: 16)-(SEQ ID NO: 16). In one embodiment, the present invention contemplates a heterodimer Acr protein comprising at least two different sequences selected from the group including, but not limited to, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and/or SEQ ID NO: 16.

In one embodiment, the present invention contemplates a heterodimer Acr protein selected from the group including, but not limited to, (SEQ ID NO: 2)-(SEQ ID NO: 4), (SEQ ID NO: 2)-(SEQ ID NO: 6), (SEQ ID NO: 2)-(SEQ ID NO: 8), (SEQ ID NO: 2)-(SEQ ID NO: 10), (SEQ ID NO: 2)-(SEQ ID NO: 12), (SEQ ID NO: 2)-(SEQ ID NO: 14), and/or (SEQ ID NO: 2)- (SEQ ID NO: 16).

In one embodiment, the present invention contemplates a heterodimer Acr protein selected from the group including, but not limited to, (SEQ ID NO: 4)-(SEQ ID NO: 6), (SEQ ID NO: 4)-(SEQ ID NO: 8), (SEQ ID NO: 4)-(SEQ ID NO: 10), (SEQ ID NO: 4)-(SEQ ID NO: 12), (SEQ ID NO: 4)-(SEQ ID NO: 14), and/or (SEQ ID NO: 4)-(SEQ ID NO: 16).

In one embodiment, the present invention contemplates a heterodimer Acr protein selected from the group including, but not limited to, (SEQ ID NO: 6)-(SEQ ID NO: 8), (SEQ ID NO: 6)-(SEQ ID NO: 10), (SEQ ID NO: 6)-(SEQ ID NO: 12), (SEQ ID NO: 6)-(SEQ ID NO: 14), and/or (SEQ ID NO: 6)-(SEQ ID NO: 16).

In one embodiment, the present invention contemplates a heterodimer Acr protein selected from the group including, but not limited to, (SEQ ID NO: 8)-(SEQ ID NO: 10), (SEQ ID NO: 8)-(SEQ ID NO: 12), (SEQ ID NO: 8)-(SEQ ID NO: 14), and/or (SEQ ID NO: 8)-(SEQ ID NO: 16).

In one embodiment, the present invention contemplates a heterodimer Acr protein selected from the group including, but not limited to, (SEQ ID NO: 10)-(SEQ ID NO: 12), (SEQ ID NO: 10)-(SEQ ID NO: 14), and/or (SEQ ID NO: 10)-(SEQ ID NO: 16).

In one embodiment, the present invention contemplates a heterodimer Acr protein selected from the group including, but not limited to, (SEQ ID NO: 12)-(SEQ ID NO: 14), and/or (SEQ ID NO: 12)-(SEQ ID NO: 16).

In one embodiment, the present invention contemplates a heterodimer Acr protein of (SEQ ID NO:14)-(SEQ ID NO:16).

The data presented herein demonstrate a direct binding of several Acrs to NmeCas9, as well as data suggesting that Acr1Nm and Acr2Nm can simultaneously bind to NmeCas9. For example, E. coli lysates containing untagged, overexpressed anti-CRISPR proteins were incubated with Ni-NTA resin alone (-) or Ni-NTA resin with 6xHis-tagged NmeCas9 bound (+) and the resultant elution fractions were analyzed by SDS-PAGE followed by Coomassie staining. See Figure 16. A direct interaction between Acr1Nm and Acr2Nnm with NmeCas9 was also shown using purified, untagged anti-CRISPR proteins mixed with Ni-NTA resin alone (-) or Ni-NTA resin with 6xHis-tagged NmeCas9 bound (+), with the input and resultant elution fractions analyzed by Coomassie-stained SDS-PAGE. See Figure 17.

Although it is not necessary to understand the mechanism of an invention, it is believed that a fusion protein comprising the active binding sites of Acr1Nm and Acr2Nm would act synergistically to provide improved gene editing than would be expected from either Acr alone or a mere additive function of being used simultaneously as separate proteins. This is because Acr1Nm and Acr2Nm are shown herein to simultaneously bind to NmeCas9. Ni-NTA resin with 6xHis-NmeCas9 bound was incubated with purified, untagged Acr2Nm followed by Acr1Nm, or vice versa. The amount of each Acr eluted with NmeCas9 was not decreased when NmeCas9 was pre-bound with the other Acr. See Figure 18. Therefore, this data suggests that Acr1Nm and Acr2Nm do not compete to bind NmeCas9 and that they bind distinct, non-overlapping sites on the NmeCas9 protein. Although it is not necessary to understand the mechanism of an invention, it is believed that these data raise the possibility that a combination of Acr1Nm and Acr2Nm would have a synergistic effect on NmeCas9 activity than either Acr alone.

C. Acr Fusion/Adduct Proteins

In one embodiment, the present invention contemplates an Acr fusion protein. In one embodiment, the fusion protein comprises a nuclear localization sequence. In one embodiment, the fusion protein comprises a epitope sequence tag. In one embodiment, the epitope sequence tag is FLAG (e.g., DYKDDDDK).

The data presented here demonstrates that FLAG-tagged Acr proteins have similar gene editing inhibitory activity as an Acr protein without a FLAG tag. For example, a titration of FLAG-tagged Acr proteins were assayed with the negative control Acr88-32 protein. The data further demonstrated similar activity whether the FLAG tag was attached to either the C-terminal or N-terminal end. For example, the gene editing of Acr1Bo was similar when attached at either the C-terminal or N-terminal end. See, Figure 11A and Figure 11B. Similar data was shown for Acr1Nm, Acr2Nm and Acr3Nm. See, Figures 12A/B, 13A/B and 14A/B, respectively. These data are quantitated and summarized. See, Figures 15A -15D.

D. Acr Mutated Proteins

In one embodiment, the present invention contemplates a mutated Acr protein. In one embodiment, the Acr protein comprises at least one non-wild type amino acid residue. In one embodiment, the Acr protein comprises at least two non-wild type amino acid residues. In one embodiment, the Acr protein comprises at least three non-wild type amino acid residues. In one embodment the non-wild type amino acid residue represents an AA ^WT → Ala mutation. In one embodiment, the non-wild type amino acid residue represents a Cys→ Arg mutation. In one embodiment, the non-wild type amino acid residue represent a Phe→ Ser mutation. III. Methods Of Using Type II Cas9 Inhibitor Compositions

In one embodiment, the present invention contemplates a method for controlling Cas9 genome editing and/or genome binding that provides a limitation on Cas9 activity. In one embodiment, the controlling is a limitation on Cas9 activity duration. In one embodiment, the controlling is a limitation on Cas9 activity efficiency. In one embodiment, the controlling is a limitation on Cas9 activity spatial extent. In one embodiment, the controlling is a limitation on Cas9 activity tissue specificity. In one embodiment, the controlling is a limitation on Cas9 activity environmental condition. A. Biological Therapeutics

In one embodiment, the present invention contemplates a method for treating and/or preventing disease. In one embodiment, the disease is a mammalian disease. In one embodiment, the mammalian disease is a human disease. In one embodiment, the mammalian disease is a livestock disease. In one embodiment, the disease is an avian disease. In one embodiment, the disease is an agricultural product disease.

1. Minimization Of Off-Target Effects

In one embodiment, the present invention contemplates a method of controlling Cas9 activity duration. For example, Cas9/sgRNA could be delivered into a cell, allowed to function for a predetermined duration that was shown empirically to yield efficient on-target editing, and then inactivated through the delivery of the Acr protein. 2. Control Of Spatial Activity

In one embodiment, the present invention contemplates a method of controlling Cas9 spatial activity providing a Cas9 having at least two tissue or cell type specific activities and at least one Acr inhibitor selective for at least one of the plurality of tissue or cell type specific activities. Although it is not necessary to understand the mechanism of an invention, it is believed that Cas9 spatial control by using Acr inhibitors would permit administration of a Cas9 having multiple tissue or cell type specific activities, where simultaneous administration of Acrs selective for unwanted tissue/cell type activity would result in Cas9 activity on only a selected subset of possible cell types or tissues.

B. Gene Drives

In one embodiment, the present invention contemplates a method for controlling Cas9- based―gene drives‖ to modify the genomes of entire populations of organisms for potentially beneficial purposes. Examples include, but are not limited to: i) preventing the spread of genes in insect vectors of infectious disease (malaria, dengue, etc.); ii) preventing the ability of a general population to act as a host for a pathogen; or iii) rendering a specific population sub- fertile or even sterile.

However, uncontrolled gene drives have the potential for negative unintended

consequences of ecosystem-wide genetic modification. anti-CRISPR proteins could possibly be used as an―antidote‖ that could slow the further spread of an―out-of-control‖ gene drive, in the event that negative unintended consequences become apparent. For instance, if a Cas9 gene drive is engineered into e.g. the mosquito Aedes aegypti, another population of A. aegypti could be generated that expresses the Acr protein, and held in reserve for later release into the same population, if needed. The Acr-expressing population would be gene drive-resistant. IV. Acr Proteins As CRISPR-Cas9“Off-Switches”

Although it is not necessary to understand the mechanism of an invention, it is believed that CRISPR-Cas9 technology would be enhanced by an ability to inhibit Cas9 function spatially, temporally, or conditionally. As discussed above, small proteins encoded by bacteriophages have been shown to inhibit the CRISPR-Cas systems of host bacteria. These ―anti- CRISPRs‖ were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. In some embodiments, the present invention contemplates a plurality of Cas9 inhibitors. For example, the data presented herein suggest at least three distinct families of anti-CRISPR proteins that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. As shown, these anti-CRISPR proteins bind directly to N. meningitidis Cas9 (NmeCas9), and can be used as potent inhibitors of genome editing, for example, in human cells. These anti-CRISPR proteins are the first known―off-switches‖ for CRISPR-Cas9 activity, and they provide a genetically encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes.

In one embodiment, the present invention contemplates a method comprising CRISPR- Cas9 mediated genome editing for clinical therapeutics. For example, Cas9 is a nuclease that can be programmed with a guide RNA molecule to cut nearly any desired DNA sequence (Gasiunas et al., 2012; Jinek et al., 2012), enabling mutagenesis or editing at the site of cleavage (Cho et al., 2013; Cong et al., 2013; Hwang et al., 2013; Jiang et al., 2013; Jinek et al., 2013; Mali et al., 2013). This RNA-guided DNA editing technology has been suggested for development to provide personalized gene therapy to correct inherited disease, for sequence- specific targeting of pathogens to treat infectious disease, and many other applications (Bikard et al., 2014; Ebina et al., 2013; Gomaa et al., 2014; Kaminski et al., 2016; Ousterout et al., 2015; Wu et al., 2013; Yin et al., 2014).

Although the utility of Cas9 DNA targeting is widely acknowledged, there are currently limited means to exert control over Cas9 activity once it has been activated or delivered, leading to practical difficulties and safety concerns that are of importance in a clinical setting. For example, off-target effects (cleavage and mutation at unintended, near-cognate genomic sites) are exacerbated by excessive or prolonged Cas9 activity (Fu et al., 2014; Hsu et al., 2013;

Pattanayak et al., 2013). Many potential therapeutic applications of CRISPR-Cas9 require editing at specific target tissues. Consequently, collateral Cas9 activity in ancillary tissues is at best useless and at worst a safety risk. For example, when zygotic injections of CRISPR Cas9 components are used to generate mutant animals, Cas9 activity after the initial rounds of mitosis can give rise to mosaic genotypes (Wang et al., 2013; Yen et al., 2014). Alternately, precise Cas9 gene editing involves homology-dependent repair (HDR). Because HDR pathways are suppressed during the G1 phase of the cell cycle, Cas9 activity during G1 increases the background of undesired imprecise edits. (Orthwein et al., 2015)) Recently, CRISPR-Cas9 gene drives (supra) have been developed, in part to advance the long-term goal of eradicating disease vectors such as mosquitos (Gantz et al., 2015; Hammond et al., 2016). A danger of this approach is that gene drives, once introduced into the environment, could be difficult to restrain, and could have unpredictable ecological consequences. Based on these and other considerations, the performance and safety of CRISPR-Cas9 applications could be greatly improved if Cas9 activity could be more effectively controlled. Several groups have devised methods to activate CRISPR Cas9 genome editing in response to specific cues, including light-inducible and drug-inducible Cas9 activity. (Nihongaki et al., 2015; Nunez et al., 2016; Wright et al., 2015). However, a robust, specific, and genetically-encodable―off-switch‖ for Cas9 activity has not yet been identified.

In one embodiment, the present invention contemplates CRISPR-Cas9 technologies that are derived from a type II CRISPR-Cas9 adaptive immune systems of bacteria. In one embodiment, a type II CRISPR-Cas9 complex can target and destroy foreign DNA entities such as bacteriophages (phages) and plasmids (Barrangou et al., 2007; Deltcheva et al., 2011).

Although the Cas9 ortholog from Streptococcus pyogenes strain SF370 (SpyCas9, subtype II-A) is commonly used, type II CRISPR-Cas systems from several other bacterial species have also been adapted for eukaryotic genome editing (Makarova et al., 2015); Cong et al., 2013; Esvelt et al., 2013; Hirano et al., 2016; Hou et al., 2013; Lee et al., 2016; Muller et al., 2016; Ran et al., 2015). For example, a Cas9 from Neisseria meningitidis (NmeCas9; subtype II-C) may be an effective tool for human genome editing (Makarova et al., 2015; Esvelt et al., 2013; Hou et al., 2013; Lee et al., 2016). One advantage of NmeCas9 is because it prone to off-target effects (Esvelt et al., 2013; Hou et al., 2013; Lee et al., 2016). Alternatively a―Dead‖ NmeCas9 (dNmeCas9), in which nuclease active-site residues have been mutated, has also proven to be an effective, specific RNA-guided genome binding platform (Esvelt et al., 2013; Hilton et al., 2015; Kearns et al., 2015; Ma et al., 2015b). Similarly, dSpyCas9 and other nuclease-inactivated orthologs may also be useful in the presently disclosed invention. (Dominguez et al., 2016;

Wang et al., 2016)).

A. Neisseria Meningitidis Anti-CRISPR Proteins

Preliminary studies characterized a conserved feature of anti-CRISPR (acr) genes as a downstream gene encoding a putative transcriptional regulator. For example, two distinct families were identified having helix-turn-helix (HTH) containing anti-CRISPR associated (Aca) proteins, which are referred to herein as Aca1 and Aca2. Identification of genes encoding Aca proteins in diverse bacterial species are also represented in five other families of type I-F acr genes encoded directly upstream of the aca genes. (Pawluk et al., 2016).

In one embodiment, the present invention contemplates a plurality of genes encoding inhibitors of type II CRISPR-Cas systems. In one embodiment, type II acrs are located upstream of aca genes in MGEs within species bearing type II systems. For example, an anti-CRISPR gene in a Brackiella oedipodis putative encodes a 91-residue hypothetical protein (accession WP_028357638.1) lying directly upstream of an aca2 gene. Figure 19A. This putative anti- CRISPR possessed several orthologs encoded in MGEs of diverse Proteobacteria, and a distant, putative ortholog in a Firmicute, Fenollaria massiliensis. Figure 20.

The data suggest that the most frequently observed homologs of the CRISPR-Cas system among these species was a type II-C homolog. In one embodiment, the present invention contemplates an anti-CRISPR family that inhibits the activity of one or more representative type II-C Cas9 orthologs.

N. meningitidis strain 8013 harbors a type II-C CRISPR-Cas system (Zhang et al., 2013; Zhang et al., 2015). Further, strains of N. meningitidis are among the genomes that contain an MGE encoding a member of this putative anti-CRISPR family. Figure 19A, Figure 20.

Consequently, NmeCas9 was used to establish proof of principle for Type II Cas9 inhibitor proteins. See, Table 1 and Table 2.

Table 1: Representative Nuclotide And Amino Acid Sequences of Type II NmeCa9 Inhibitor Proteins

Candidate type II-C anti-CRISPR proteins from B. oedipodis and N. meningitidis genes to inhibit type II-C CRISPR-Cas activity was measured in their native context, using a previously described natural transformation assay in N. meningitidis 8013. Figure 19A; Zhang et al., 2013; Zhang et al., 2015. In this assay, the transformation frequency of a plasmid bearing a CRISPR targeted protospacer sequence was compared to that of a control plasmid lacking the protospacer. A wild-type strain, as well as isogenic derivatives with an integrated, empty nics (e.g., an Neisseria intergenic complementation site), and either of the two candidate anti-CRISPR proteins were driven by a N. meningitidis cas9 promoter. Figure 19B. In wild-type cells and the empty-vector control, robust type II-C CRISPR-Cas activity resulted in a≥ 10 ⁴ -fold decrease in the transformation frequency of CRISPR-targeted DNA. Strikingly, expression of the putative anti-CRISPR proteins resulted in equal transformation frequencies when targeted or untargeted DNA was used, reflecting a lack of CRISPR interference. Figure 19C.

These data showed that the type II-C CRISPR-Cas system of N. meningitidis was inhibited by proteins encoded by at least two putative anti-CRISPR genes, which are named herein acrIIC1 _Boe and acrIIC1 _Nme . Although acrIIC1 _Boe has presumably evolved to inhibit the Cas9 ortholog found in B. oedipodis (BoeCas9), NmeCas9 is 47% identical to BoeCas9, suggesting that this similarity is sufficient to account for the observed cross-species inhibition. For the most part, the above identified acrIIC1 orthologs, except acrIIC1 _Boe , were not found adjacent to aca1 or aca2 genes, but were instead encoded upstream of genes encoding distinct HTH-containing proteins. Figure 19, Figure 20.

In one embodiment, the present invention contemplates a plurality of aca genes that encode a plurality of anti-CRISPR proteins. For example, two distinct genes were cloned that were located in a putative N. meningitidis prophage immediately upstream of the candidate aca3 gene. These genes also displayed robust anti-CRISPR activity and are referred to herein as acrIIC2 _Nme and acrIIC3 _Nme . Figures 19B and 19C; Table 1 and Table 2. These results demonstrate the existence of anti-CRISPR genes that are active against a type II CRISPR-Cas system.

B. Direct Interaction Of Type II-C anti-CRISPR Proteins With NmeCas9 A direct interaction of type II-C anti-CRISPR proteins with NmeCas9 was determined by mixing purified, untagged anti-CRISPR proteins with purified, 6xHis-tagged NmeCas9 protein (preloaded with coexpressed sgRNA) and conducting nickel affinity chromatography to assess whether the anti-CRISPR proteins directly bound NmeCas9 in vitro. It was found that

AcrIIC1Nme, AcrIIC2Nme, and AcrIIC3Nme were all retained on the nickel column, reflecting an association with NmeCas9. By contrast, a previously identified type I anti-CRISPR protein (AcrE2; (Pawluk et al., 2014)) did not associate with NmeCas9, and the anti-CRISPR proteins did not bind significantly to AnaCas9. Figures 21B and 22. AnaCas9 is a distantly related type II-C Cas9 homolog with ~20% sequence identity to NmeCas9. Jinek et al., 2014; Ma et al., 2015a. These data demonstrate that the anti-CRISPR proteins disclosed herein specifically bind to NmeCas9.

To assess the effect of the anti-CRISPR proteins on Cas9 enzymatic activity, in vitro DNA cleavage assays were performed. (Zhang et al., 2015) When purified NmeCas9 was loaded with in vitro transcribed sgRNA and then mixed with target DNA, robust and specific cleavage was observed. Figure 23. Cleavage was unaffected by prior incubation of NmeCas9 with increasing amounts of the control, type I-specific anti-CRISPR, AcrE2. In contrast, addition of the N. meningitidis anti-CRISPR proteins to these reactions resulted in inhibition of NmeCas9- catalyzed cleavage in a dose-dependent manner. Approximately 50% cleavage inhibition resulted when the anti-CRISPR proteins were added at a 1:1 molar ratio, and complete inhibition was seen at a 5:1 anti-CRISPR:NmeCas9 ratio. Figure 23. The DNA cleavage activity of S. pyogenes Cas9 (SpyCas9), which is the most commonly used Cas9 for genome editing, was not affected by addition of any of the anti-CRISPR proteins (Figure 23, lower panel). This result was expected because SpyCas9 belongs to the type II-A CRISPR-Cas type and is very distantly related to NmeCas9. Overall, these in vitro data clearly demonstrate that these anti-CRISPR proteins directly bind to and specifically inhibit the DNA cleavage activity of NmeCas9. The inhibitory effects of anti-CRISPR proteins on NmeCas9 in its sgRNA-loaded form imply that the natural protective functions of the anti-CRISPR proteins require inhibition of crRNA/tracrRNA- loaded NmeCas9 that is already present in the host cell at the time of phage infection.

C. Anti-CRISPR proteins Inhibit NmeCas9 Genome Editing

In one embodiment, the present invention contemplates a plurality of direct anti-CRISPR protein inhibitors of NmeCas9 activity that are off-switches for CRISPR-Cas9 genome editing in mammalian cells. For example, HEK293T cells were co-transfected with three plasmids: one expressing NmeCas9, one expressing a genome-targeting sgRNA, and one expressing an anti- CRISPR. Genome editing efficiency was determined using an established T7 endonuclease 1 (T7E1)-based protocol. Strikingly, it was found that each of the anti-CRISPR proteins greatly decreased the ability of NmeCas9 to create genome lesions in cultured human cells. Figure 24, Figure 25, Figure 26 and Table 3.

In one embodiment, the present invention contemplates anti-CRISPR proteins that are less than approximately 14 kDa. Although it is not necessary to understand the mechanism of an invention it is believed that these anti-CRISPR proteins are small enough to diffuse freely through nuclear pores such that they can inhibit NmeCas9 genome editing even without an appended, heterologous nuclear localization sequence (NLS). These plasmid titration experiments demonstrated that at least three anti-CRISPR families could completely inhibit type II Cas9 gene editing (e.g., 100% inhibition), with

AcrIIC3Nme appearing to be the most potent. However, gene editing was inhibited at least 70%, preferably 75% and most preferably 90% or greater. Figure 26. The superior potency of

AcrIIC3Nme anti-CRISPR activity in mammalian cells is noteworthy given that it was slightly less effective at inhibiting transformation interference in meningococcal cells. Figure 19C. The variations in activities of these anti-CRISPR proteins in mammalian cells are likely due to differences in expression or stability as they all displayed similar inhibitory activities in vitro. Figure 23. Consistent with other in vitro results, the anti-CRISPR proteins had no effect on editing mediated by SpyCas9 targeting the same genomic site. Figures 24 and 25. In addition, type I-E anti-CRISPR AcrE2 had no significant inhibitory effect in any of these experiments. In no instance was any sign of cellular toxicity observed by any anti-CRISPR protein. In one embodiment, the present invention contemplates a plurality of anti-CRISPR proteins that precisely control Cas9-mediated genome editing by means of inhibition.

D. AcrIIC3Nme Prevents dNmeCas9 Genome Binding

―Dead‖ Cas9 (dCas9) orthologs, including dNmeCas9 have been reported to be useful for RNA-guided DNA binding without Cas9-catalyzed DNA cleavage. Esvelt et al., 2013; Hilton et al., 2015; 228 Kearns et al., 2015; Ma et al., 2015b. Although it is not necessary to understand the mechanism of an invention, it is believed that dCas9 proteins have a wide range of domains and functionalities that can be fused or tethered to the DNA-bound dCas9/sgRNA complex. Dominguez et al., 2016; Wang et al., 2016.

As shown above, anti-CRISPR inhibition of sgRNA-guided NmeCas9 DNA cleavage and genome editing could reflect either inhibition upstream of stable R-loop formation, or inhibition of NmeCas9 catalytic activation after stable R-loop formation. Figures 23 and 24. In the former case, the anti-CRISPR could be used as an off-switch not only for genome editing, but also for dNmeCas9 DNA binding applications such as CRISPRi and CRISPRa. Dominguez et al., 2016; Wang et al., 2016.

To determine whether a genome editing inhibitor (e.g., AcrIIC3Nme) can prevent stable DNA binding by dNmeCas9 in mammalian cells, a previously developed system was used in which superfolder (sf) GFP-labeled dNmeCas9 and mCherry-labeled dSpyCas9 are

simultaneously colocalized to telomeric loci by cognate sgRNAs upon co-transfection of their expression plasmids in U2OS cells. Figure 27A; Ma et al., 2015b. Colocalization of telomeric dNmeCas9-(sfGFP) ₃ and dSpyCas9-(mCherry) ₃ foci was observed as long as both of the telomere-directed sgRNAs were included as the two dCas9 orthologs. Figures 27B-D. A co- transfected, mTagBFP2-marked plasmid also carrying an anti-CRISPR expression cassette was also tested. Figure 27A, bottom. AcrE2 had no effect on telomeric colocalization of dNmeCas9- (sfGFP) ₃ and dSpyCas9-(mCherry) ₃ . Figure 27E. In contrast, co-expression of AcrIIC3Nme prevented the co-localization of dNmeCas9-(sfGFP) ₃ with the dSpyCas9-(mCherry) ₃ telomeric foci. Figure 27F.

Telomeric dNmeCas9-(sfGFP) ₃ foci were observed in 94% (31 out of 33) of cells in the absence of any Acr protein, and 88% (31 out of 37) of cells in the presence of the negative control AcrE2 protein. By contrast, 0% of cells (0 out of 46) exhibited dNmeCas9-(sfGFP)3 telomeric foci when AcrIIC3Nme was coexpressed. These results confirm the robust inhibitory effect of AcrIIC3Nme on stable, sgRNA-programmed DNA binding by dNmeCas9, and indicate that they can be used as a potent off-switch not only for NmeCas9 genome editing, but also for dNmeCas9-based applications in mammalian cells.

E. Global Inhibition Of Type II-C CRISPR-Cas9 By Anti-CRISPR Proteins CRISPR-Cas systems may be divided into at least two broad classes, each encompassing several types and many subtypes. For example, Class 1 systems employ multi-subunit surveillance complexes, whereas Class 2 systems have single, large effector proteins like Cas9. Makarova et al., 2015; Figure 28/6A. Preliminary data on anti-CRISPR proteins acting on type I systems (i.e., for example belonging to Class 1) suggest that each anti-CRISPR protein acts on a particular range of systems within one subtype due to the specificity of protein-protein interactions between the anti-CRISPR and Cas proteins. Bondy-Denomy et al., 2015; Bondy- Denomy et al., 2013; Pawluk et al., 2014; Pawluk et al., 2016. Although it is not necessary to understand the mechanism of an invention, it is believed that an anti-CRISPR gene will likely be selected for if it inhibits the CRISPR-Cas system of the bacterium in which it is found as these genes are almost always located on MGEs that have successfully invaded a host. This belief was used to accurately predict that some anti-CRISPR proteins described here might block type II-C CRISPR-Cas system of N. meningitidis. A potential general impact of anti-CRISPR activity on type II CRISPR-Cas systems can be visualized using a phylogenetic tree of Cas9 showing their relationships as direct binding targets of the type II-C anti-CRISPR proteins. Figure 28B. Bacterial genera in which known type II-C anti-CRISPR homologs are encoded are indicated in red. From this analysis, based on the phylogenetic breadth spanned by the anti-CRISPR orthologs, it can be proposed that greater than half of the type II-C CRISPR-Cas systems may be susceptible to three of the anti-CRISPR families disclosed herein. These data, combined with preliminary studies of type I-F CRISPR-Cas systems and their cognate anti-CRISPR proteins, suggests that even the relatively small number of anti-CRISPR gene families discovered to date can have a broad impact on CRISPR-Cas systems in bacteria. As anti-CRISPR proteins have been reported to inhibit both Class 1 and Class 2 CRISPR-Cas systems, the methods described herein would be expected to identify additional anti-CRISPR proteins that might be capable of inhibiting all types and subtypes of CRISPR-Cas systems.

The data presented herein is the first to describe the existence of three different families of type II-C anti-CRISPR proteins. In one embodiment, the present invention contemplates anti- CRISPR proteins that block genome editing by NmeCas9 in cultured human cells. Although it is not necessary to understand the mechanism of an invention, it is believed that genetically encoded Cas9 inhibitors provide a means to spatially, temporally, or conditionally control Cas9 activity, thereby potentially allowing tissue-, cell cycle stage-, developmental stage-, or stimulus- specific inactivation of genome editing.

It is generally believed that target site precision and tissue specificity play a role when considering clinical CRISPR-Cas9 applications of gene therapy, where prolonged or

misexpressed nuclease activity may exacerbate undesirable off-target effects. In one

embodiment, the present invention contemplates an effective Cas9 off-switch that ameliorates off-target effects through expression or delivery strategies that enable anti-CRISPR proteins to accumulate whenever or wherever editing activity is unwanted. In one embodiment, the present invention contemplates methods comprising anti-CRISPR proteins for regulating a gene drive with CRISPR-Cas9 to force inheritance of desired alleles (e.g. in insect populations), possession of a functioning―off-switch‖ may provide a useful security or containment measure to avert unintended adverse consequences.

The data presented herein also show that anti-CRISPR proteins may bind directly to the NmeCas9/sgRNA complex and inhibit in vitro DNA cleavage. Given the completely unrelated sequences of these anti-CRISPR proteins, it may be expected that the anti-CRISPR proteins may abrogate activity through different mechanisms. This mechanism was the case for previously reported Type I-F anti-CRISPR proteins. Bondy-Denomy et al., 2015. As the data has determined that AcrIIC3Nme prevents stable genomic localization of sgRNA-loaded dNmeCas9 in mammalian cells, these proteins can be used as an off-switch for dNmeCas9-based

applications. Alternatively, it remains possible that other anti-CRISPR proteins might allow NmeCas9 DNA-binding activity but prevent catalytic activation. If so, this mechanims would effectively create an NmeCas9 complex with a utility for modulation of transcription, similar to a type I-F anti-CRISPR protein. Dominguez et al., 2016; Wang et al., 2016; Bondy-Denomy et al., 2015.

It should be noted that CRISPR-Cas systems are present in approximately half of sequenced prokaryotic genomes and are widespread across diverse bacterial and archaeal lineages. The extreme diversity in, and purifying selective pressure on, CRISPR-Cas systems, combined with the co-occurrence of several different CRISPR-Cas system types in many genomes, is indicative of a dynamic co-evolutionary battle for survival between prokaryotes and parasitic MGEs. Makarova et al., 2015; Takeuchi et al., 2012. CRISPR-Cas systems are expected to pose a significant challenge to the process of horizontal gene transfer, especially given their ability to acquire heritable immunity against newly encountered threats and to upgrade their arsenal through new spacer acquisition. Barrangou et al., 2007; Fineran et al., 2014; Richter et al., 2014. However, recent studies have shown that the presence of a CRISPR Cas system does not correlate with lower levels of HGT over evolutionary timescales, or with a lower number of acquired prophage elements. Gophna et al., 2015; Touchon et al., 2016. We propose that widespread MGE-encoded anti-CRISPR proteins could reconcile this paradox. Also from an evolutionary perspective, it is noted that Cas9 from type II-A systems play a role not only for the interference function of existing spacers, but also for the adaptive acquisition of new spacers. Heler et al., 2015; Wei et al., 2015. If this adapation role of type II-A Cas9 extends to type II-C systems, as seems likely, then Cas9-associating anti-CRISPR proteins may prevent the acquisition of new spacers in response to ongoing invasions.

A recent in vitro evolution study showed that the only way for phages to escape

CRISPR338 mediated extinction is by the expression of an anti-CRISPR gene (van Houte et al., 2016). In strong accordance with the Red Queen theory, it has been reported that a total of seventeen distinct anti-CRISPR protein families are widespread among Proteobacteria, each inhibiting either type I-E, I-F, or II-C systems. Bondy-Denomy et al., 2013; Pawluk et al., 2014; Pawluk et al., 2016. The fact that anti-CRISPR proteins have evolved to inhibit both Class 1 and Class 2 CRISPR-Cas systems strongly suggests that they exist for other CRISPR-Cas types as well. Figure 28A. It may be suggested that anti-CRISPR activity can have a large impact on CRISPR-Cas systems across prokaryotes and on horizontal gene transfer. V. Kits

In one embodiment, the present invention contemplates a kit comprising an Acr protein. In one embodiment, the kits may be used for biological therapeutics. In one embodiment the kits may be used for gene drives. In one embodiment, the kit may be used for cell line manipulation. In one embodiment, the kit may be used for drug target validation. VI. Pharmaceutical Formulations

The present invention further provides pharmaceutical compositions (e.g., comprising the compounds described above). The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal or intraventricular

administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.

Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (U.S. Pat. No.5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 97/30731), also enhance the cellular uptake of

oligonucleotides.

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The

administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC ₅₀ s found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 μg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years. VI. Composition Delivery Systems

The present invention contemplates several composition delivery systems that provide for roughly uniform distribution, have controllable rates of release of their components (e.g., vectors, proteins, nucleic acids, drugs etc.). A variety of different media are described below that are useful in creating composition delivery systems. It is not intended that any one medium or carrier is limiting to the present invention. Note that any medium or carrier may be combined with another medium or carrier; for example, in one embodiment a polymer microparticle carrier attached to a compound may be combined with a gel medium.

Carriers or mediums contemplated by this invention comprise a material selected from the group comprising gelatin, collagen, cellulose esters, dextran sulfate, pentosan polysulfate, chitin, saccharides, albumin, fibrin sealants, synthetic polyvinyl pyrrolidone, polyethylene oxide, polypropylene oxide, block polymers of polyethylene oxide and polypropylene oxide, polyethylene glycol, acrylates, acrylamides, methacrylates including, but not limited to, 2- hydroxyethyl methacrylate, poly(ortho esters), cyanoacrylates, gelatin-resorcin-aldehyde type bioadhesives, polyacrylic acid and copolymers and block copolymers thereof.

One embodiment of the present invention contemplates a composition delivery system comprising therapeutic agents as described herein. Microparticles

One embodiment of the present invention contemplates a medium comprising a microparticle. Preferably, microparticles comprise liposomes, nanoparticles, microspheres, nanospheres, microcapsules, and nanocapsules. Preferably, some microparticles contemplated by the present invention comprise poly(lactide-co-glycolide), aliphatic polyesters including, but not limited to, poly-glycolic acid and poly-lactic acid, hyaluronic acid, modified polysacchrides, chitosan, cellulose, dextran, polyurethanes, polyacrylic acids, psuedo-poly(amino acids), polyhydroxybutrate-related copolymers, polyanhydrides, polymethylmethacrylate, poly(ethylene oxide), lecithin and phospholipids.

Liposomes

One embodiment of the present invention contemplates liposomes capable of attaching and releasing therapeutic agents described herein. Liposomes are microscopic spherical lipid bilayers surrounding an aqueous core that are made from amphiphilic molecules such as phospholipids. For example, a liposome may trap a therapeutic agent between the hydrophobic tails of the phospholipid micelle. Water soluble agents can be entrapped in the core and lipid- soluble agents can be dissolved in the shell-like bilayer. Liposomes have a special characteristic in that they enable water soluble and water insoluble chemicals to be used together in a medium without the use of surfactants or other emulsifiers. Liposomes can form spontaneously by forcefully mixing phosopholipids in aqueous media. Water soluble compounds are dissolved in an aqueous solution capable of hydrating phospholipids. Upon formation of the liposomes, therefore, these compounds are trapped within the aqueous liposomal center. The liposome wall, being a phospholipid membrane, holds fat soluble materials such as oils. Liposomes provide controlled release of incorporated compounds. In addition, liposomes can be coated with water soluble polymers, such as polyethylene glycol to increase the pharmacokinetic half-life. One embodiment of the present invention contemplates an ultra high-shear technology to refine liposome production, resulting in stable, unilamellar (single layer) liposomes having specifically designed structural characteristics. These unique properties of liposomes, allow the

simultaneous storage of normally immiscible compounds and the capability of their controlled release.

In some embodiments, the present invention contemplates cationic and anionic liposomes, as well as liposomes having neutral lipids. Preferably, cationic liposomes comprise negatively-charged materials by mixing the materials and fatty acid liposomal components and allowing them to charge-associate. Clearly, the choice of a cationic or anionic liposome depends upon the desired pH of the final liposome mixture. Examples of cationic liposomes include lipofectin, lipofectamine, and lipofectace.

One embodiment of the present invention contemplates a medium comprising liposomes that provide controlled release of at least one therapeutic agent. Preferably, liposomes that are capable of controlled release: i) are biodegradable and non-toxic; ii) carry both water and oil soluble compounds; iii) solubilize recalcitrant compounds; iv) prevent compound oxidation; v) promote protein stabilization; vi) control hydration; vii) control compound release by variations in bilayer composition such as, but not limited to, fatty acid chain length, fatty acid lipid composition, relative amounts of saturated and unsaturated fatty acids, and physical

configuration; viii) have solvent dependency; iv) have pH-dependency and v) have temperature dependency.

The compositions of liposomes are broadly categorized into two classifications.

Conventional liposomes are generally mixtures of stabilized natural lecithin (PC) that may comprise synthetic identical-chain phospholipids that may or may not contain glycolipids.

Special liposomes may comprise: i) bipolar fatty acids; ii) the ability to attach antibodies for tissue-targeted therapies; iii) coated with materials such as, but not limited to lipoprotein and carbohydrate; iv) multiple encapsulation and v) emulsion compatibility. Liposomes may be easily made in the laboratory by methods such as, but not limited to, sonication and vibration. Alternatively, compound-delivery liposomes are commercially available. For example, Collaborative Laboratories, Inc. are known to manufacture custom designed liposomes for specific delivery requirements.

Microspheres, Microparticles And Microcapsules

Microspheres and microcapsules are useful due to their ability to maintain a generally uniform distribution, provide stable controlled compound release and are economical to produce and dispense. Preferably, an associated delivery gel or the compound-impregnated gel is clear or, alternatively, said gel is colored for easy visualization by medical personnel.

Microspheres are obtainable commercially (Prolease ^® , Alkerme's: Cambridge, Mass.). For example, a freeze dried medium comprising at least one therapeutic agent is homogenized in a suitable solvent and sprayed to manufacture microspheres in the range of 20 to 90 μm.

Techniques are then followed that maintain sustained release integrity during phases of purification, encapsulation and storage. Scott et al., Improving Protein Therapeutics With Sustained Release Formulations, Nature Biotechnology, Volume 16:153-157 (1998).

Modification of the microsphere composition by the use of biodegradable polymers can provide an ability to control the rate of therapeutic agent release. Miller et al., Degradation Rates of Oral Resorbable Implants {Polylactates and Polyglycolates: Rate Modification and Changes in PLA/PGA Copolymer Ratios, J. Biomed. Mater. Res., Vol. II:711-719 (1977).

Alternatively, a sustained or controlled release microsphere preparation is prepared using an in-water drying method, where an organic solvent solution of a biodegradable polymer metal salt is first prepared. Subsequently, a dissolved or dispersed medium of a therapeutic agent is added to the biodegradable polymer metal salt solution. The weight ratio of a therapeutic agent to the biodegradable polymer metal salt may for example be about 1:100000 to about 1:1, preferably about 1:20000 to about 1:500 and more preferably about 1:10000 to about 1:500. Next, the organic solvent solution containing the biodegradable polymer metal salt and therapeutic agent is poured into an aqueous phase to prepare an oil/water emulsion. The solvent in the oil phase is then evaporated off to provide microspheres. Finally, these microspheres are then recovered, washed and lyophilized. Thereafter, the microspheres may be heated under reduced pressure to remove the residual water and organic solvent. Other methods useful in producing microspheres that are compatible with a biodegradable polymer metal salt and therapeutic agent mixture are: i) phase separation during a gradual addition of a coacervating agent; ii) an in-water drying method or phase separation method, where an antiflocculant is added to prevent particle agglomeration and iii) by a spray- drying method.

In one embodiment, the present invention contemplates a medium comprising a microsphere or microcapsule capable of delivering a controlled release of a therapeutic agent for a duration of approximately between 1 day and 6 months. In one embodiment, the microsphere or microparticle may be colored to allow the medical practitioner the ability to see the medium clearly as it is dispensed. In another embodiment, the microsphere or microcapsule may be clear. In another embodiment, the microsphere or microparticle is impregnated with a radio-opaque fluoroscopic dye.

Controlled release microcapsules may be produced by using known encapsulation techniques such as centrifugal extrusion, pan coating and air suspension. Such microspheres and/or microcapsules can be engineered to achieve desired release rates. For example,

Oliosphere ^® (Macromed) is a controlled release microsphere system. These particular microsphere's are available in uniform sizes ranging between 5 - 500 μm and composed of biocompatible and biodegradable polymers. Specific polymer compositions of a microsphere can control the therapeutic agent release rate such that custom-designed microspheres are possible, including effective management of the burst effect. ProMaxx ^® (Epic Therapeutics, Inc.) is a protein-matrix delivery system. The system is aqueous in nature and is adaptable to standard pharmaceutical delivery models. In particular, ProMaxx ^® are bioerodible protein microspheres that deliver both small and macromolecular drugs, and may be customized regarding both microsphere size and desired release characteristics.

In one embodiment, a microsphere or microparticle comprises a pH sensitive

encapsulation material that is stable at a pH less than the pH of the internal mesentery. The typical range in the internal mesentery is pH 7.6 to pH 7.2. Consequently, the microcapsules should be maintained at a pH of less than 7. However, if pH variability is expected, the pH sensitive material can be selected based on the different pH criteria needed for the dissolution of the microcapsules. The encapsulated compound, therefore, will be selected for the pH

environment in which dissolution is desired and stored in a pH preselected to maintain stability. Examples of pH sensitive material useful as encapsulants are Eudragit ^® L-100 or S-100 (Rohm GMBH), hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate phthalate, and cellulose acetate trimellitate. In one embodiment, lipids comprise the inner coating of the microcapsules. In these compositions, these lipids may be, but are not limited to, partial esters of fatty acids and hexitiol anhydrides, and edible fats such as triglycerides. Lew C. W., Controlled-Release pH Sensitive Capsule And Adhesive System And Method. United States Patent No.5,364,634 (herein incorporated by reference).

In one embodiment, the present invention contemplates a microparticle comprising a gelatin, or other polymeric cation having a similar charge density to gelatin (i.e., poly-L-lysine) and is used as a complex to form a primary microparticle. A primary microparticle is produced as a mixture of the following composition: i) Gelatin (60 bloom, type A from porcine skin), ii) chondroitin 4-sulfate (0.005% - 0.1%), iii) glutaraldehyde (25%, grade 1), and iv) 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC hydrochloride), and ultra-pure sucrose (Sigma Chemical Co., St. Louis, Mo.). The source of gelatin is not thought to be critical; it can be from bovine, porcine, human, or other animal source. Typically, the polymeric cation is between 19,000-30,000 daltons. Chondroitin sulfate is then added to the complex with sodium sulfate, or ethanol as a coacervation agent.

Following the formation of a microparticle, a therapeutic agent is directly bound to the surface of the microparticle or is indirectly attached using a "bridge" or "spacer". The amino groups of the gelatin lysine groups are easily derivatized to provide sites for direct coupling of a compound. Alternatively, spacers (i.e., linking molecules and derivatizing moieties on targeting ligands) such as avidin-biotin are also useful to indirectly couple targeting ligands to the microparticles. Stability of the microparticle is controlled by the amount of glutaraldehyde- spacer crosslinking induced by the EDC hydrochloride. A controlled release medium is also empirically determined by the final density of glutaraldehyde-spacer crosslinks.

In one embodiment, the present invention contemplates microparticles formed by spray- drying a composition comprising fibrinogen or thrombin with a therapeutic agent. Preferably, these microparticles are soluble and the selected protein (i.e., fibrinogen or thrombin) creates the walls of the microparticles. Consequently, the therapeutic agents are incorporated within, and between, the protein walls of the microparticle. Heath et al., Microparticles And Their Use In Wound Therapy. United States Patent No.6,113,948 (herein incorporated by reference).

Following the application of the microparticles to living tissue, the subsequent reaction between the fibrinogen and thrombin creates a tissue sealant thereby releasing the incorporated compound into the immediate surrounding area.

One having skill in the art will understand that the shape of the microspheres need not be exactly spherical; only as very small particles capable of being sprayed or spread into or onto a surgical site (i.e., either open or closed). In one embodiment, microparticles are comprised of a biocompatible and/or biodegradable material selected from the group consisting of polylactide, polyglycolide and copolymers of lactide/glycolide (PLGA), hyaluronic acid, modified polysaccharides and any other well known material. VII. Phage Therapy

In one embodiment, the present invention contemplates a composition comprising bacteriophage genes, a Type II Cas9/sgRNA gene complex, and a gene encoding an Acr protein. In one embodiment, the present invention contemplates a method, comprising; a) providing; i) a patient comprising an infection by a bacteria, wherein said bacteria comprises a CRISPR-Cas9 system; and ii) a bacteriophage comprising a gene encoding an Acr protein; b) administering said bacteriophage to said patient such that said bacteriophage contacts said bacteria; c) injecting said bacteria with said Acr gene under conditions that said Acr gene is expressed; and d) inhibiting said bacterial CRISPR-Cas9 system with said expressed Acr gene. In one embodiment, the bacteriophage further comprises a CRISPR-Cas9 gene complex comprising a sgRNA sequence that is capable of binding to a bacterial gene target site. In one embodiment, the bacteriophage CRISPR-Cas9 gene complex is expressed in said bacteria and edits said bacterial gene.

Although it is not necessary to understand the mechanism of an invention, it is believed that some bacteria have CRISPR-Cas systems that would inactivate the phage, therefore making conventional phage therapy extremely ineffective. In one embodiment, the method further comprises adminstering an Acr protein to inhibit a bacterial CRISPR-Cas system thereby permitting a productive phage infection, and subsequent reduction of bacterial infection symptoms. Notably, Neisseria meningitidis and the closely related Neisseria gonorrhoeae are human pathogens for which phage therapy may be extremely useful as they are rapidly acquiring antibiotic resistance. In one embodiment, the present invention contemplates a method of phage therapy on mammalian cells. Phage therapy or viral phage therapy is the therapeutic use of bacteriophages to treat pathogenic infections. Phage therapy has many potential applications in human medicine as well as dentistry, veterinary science, and agriculture. If the target host of a phage therapy treatment is not an animal the term "biocontrol" (as in phage-mediated biocontrol of bacteria) is usually employed, rather than "phage therapy".

Although the natural hosts for bacteriophages are bacteria, a growing body of data shows that phages can also interact with some populations of mammalian cells, especially with cells of the immune system. In general, these interactions include two main aspects. The first is the phage immunogenicity, that is, the capacity of phages to induce specific immune responses, in particular the generation of specific antibodies against phage antigens. The other aspect includes the immunomodulatory activity of phages, that is, the nonspecific effects of phages on different functions of major populations of immune cells involved in both innate and adaptive immune responses. These functions include, among others, phagocytosis and the respiratory burst of phagocytic cells, the production of cytokines, and the generation of antibodies against nonphage antigens. The aim of this chapter is to discuss the interactions between phages and cells of the immune system, along with their implications for phage therapy. These topics are presented based on the results of experimental studies and unique data on immunomodulatory effects found in patients with bacterial infections treated with phage preparations. Górski et al.,―Phage as a modulator of immune responses: practical implications for phage therapy‖ Adv Virus Res.

2012;83:41-71.

Bacteriophage T4 is a virus with well-known genetics, structure, and biology. Such techniques as X-ray crystallography, cryo-EM, and three-dimensional (3D) image reconstruction allowed describing its structure very precisely. The genome of this bacteriophage was completely sequenced, which opens the way for the use of many molecular techniques, such as site-specific mutagenesis, which was widely applied, e.g., in investigating the functions of some essential T4 proteins. The phage-display method, which is commonly applied in bacteriophage modifications, was successfully used to display antigens (PorA protein, VP2 protein of vvIBDV, and antigens of anthrax and HIV) on T4's capsid platform. As first studies showed, the phage- display system as well as site-specific mutagenesis may also be used to modify interactions between phage particles and mammalian cells or to obtain phages infecting species other than the host bacteria. These may be used, among others, in the constantly developing bacteriophage therapy. All manipulations of this popular bacteriophage may enable the development of vaccine technology, phage therapy, and other branches of biological and medical science. Kurzepa et al., ―Molecular modification of T4 bacteriophage proteins and its potential application - review‖ Folia Microbiol (Praha).2009;54(1):5-15. Epub 2009 Mar 29.

Bacteriophages are viruses infecting bacteria and lack ability to infect mammalian cells but promises to be a novel divergent therapeutic approach. The great versatility of the phage system has led to the development of improved phage delivery vectors. Combination therapy based on multiple phage and other pharmaceutical treatments holds great promise. The potential therapeutic phage therapy arises from its lack of natural tropism for mammalian cells, resulting in no adverse effects. Sohrab et al.,―Bacteriophage--a common divergent therapeutic approach for Alzheimer's disease and type II diabetes mellitus‖ CNS Neurol Disord Drug Targets.2014 13(3):491-500.

Advances in phage therapy encourage scientific interest in interactions of phages with human and animal organisms. This has created a need for developing tools that facilitate studies of phage circulation and deposition in tissues and cells. A new green fluorescent protein (GFP)- based method has been disclosed to administer T4 phage molecular imaging in living systems. The method employs decoration of a phage capsid with GFP fused to the N-terminus of Hoc protein by in vivo phage display. Fluorescent phages were positively assessed as regards their applicability for detection inside living mammalian cells (by phagocytosis) and tissues (filtering and retention by lymph nodes and spleen). Kaźmierczak et al.,―Molecular imaging of T4 phage in mammalian tissues and cells‖ Bacteriophage.20144(1):e28364. Epub 2014 Feb 27.

Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage), viruses that are generally known to infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. Chemical modifications of the bacteriophage capsid have been proposed to improve targeted gene delivery by phage vectors into mammalian cells. For example, a hybrid system including two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles were genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. A phage complex with cationic polymers generates positively charged phage and large aggregates show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage. Yata et al.,―Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery‖ Mol Ther Nucleic Acids.2014 Aug 12;3:e185. Experimental

Example I

Construction Of Acr Vectors

Sequences that are found to encode Acr proteins can be ordered from companies that market custom DNA sequence samples, or they can be PCR-amplified from suitable biological DNA samples. They can then be introduced into a vector (including, but not limited to, plasmids and viral vectors) by standard recombinant DNA cloning protocols (e.g. restriction endonuclease digestion and religation, or Gibson assembly). The vector commonly includes promoter sequences suitable for expression within the cells of interest. They also contain other sequences that facilitate gene expression and control [e.g., 5’ and 3’ untranslated regions, poly(A) signals, and transcription terminators]. These vectors can then be introduced into target cells using standard delivery approaches that are well known in the field. Example II

Screening For Acr Protein Candidates

Many other acr gene candidates can be identified by their genetic association with aca genes or putative aca genes. Acr open reading frames can be cloned directly from the species of origin, or, alternatively, the gene sequence can be synthesized by a company. There are several ways to screen for Acr activity against the Type II system.1) test for inhibition of NmeCas9 activity in its native context, i.e. in Neisseria meninigitidis by using a previously described assay measuring the transformation efficiency of CRISPR-Cas9 targeted DNA; 2) test for inhibition of NmeCas9 activity in a heterologous E. coli system whereby NmeCas9 is programmed to target E. coli bacteriophage (e.g. lambda phage); 3) test for inhibition of NmeCas9 activity in human cell culture using the T7E1 assays described herein. Example III

Acr Administration To Control the Spread of Gene Drives Cas9 is currently being contemplated for use in gene drives, which are engineered genetic elements that can spread rapidly through a natural population. If that natural population is an undesirable one, e.g. an insect that transmits a pathogen such as the malaria parasite or dengue virus, then the gene drive could include elements that induce death or sterility to any insect that acquires the gene drive. Although the potential of this approach to control insect- borne diseases is widely recognized, there are many concerns about their safety and the potential for unintended adverse consequences (predictable or not) upon release into the natural environment. Part of this concern arises from the possibility that spread of the gene drive could be difficult or impossible to stop once it is released. A gene drive could be envisioned that uses a Type II-C Cas9 enzyme, e.g. NmeCas9, to support its propagation. Prior to release of the gene drive, a separate insect population could be generated that is engineered to express the Acr protein, rendering them resistant to the gene drive. If adverse consequences of gene drive propagation become apparent, the Acr-expressing insect population could be released to slow the propagation of the gene drive through the population. Example IV

Acr Administration For Biological Therapeutics

NmeCas9 and its sgRNA could be encoded in a vector and then introduced into the cells of a patient for purposes of therapeutic genome editing. Once the intended editing has occurred, further NmeCas9 activity is undesirable: because the intended editing site has been altered, the only potential editing sites left are unintended ones. DNA cleavage at unintended sites could be harmful to the patient (for example, they could induce oncogenic chromosome translocations). After the intended editing event has occurred, a vector encoding the Acr protein could be introduced into the patient’s cells to inhibit NmeCas9 from that point forward, preventing these unintended DNA cleavage events from occurring. Example V

Creation Of Acr Fusion Proteins

Two Acrs that are found to bind different areas on the surface of the Cas9 protein may be fused together to provide potentially synergistic effects on Cas9 inhibition. Non-competitive, simultaneous binding can be detected using in vitro experiments where the Cas9 protein is saturated with one Acr, and then a second Acr is added and its binding is assessed. If the second Acr can bind to Cas9 without displacing the first Acr, then their binding must be non-competitive and they bind to non-overlapping areas on the Cas9 protein. This is conceivable because the Cas9 protein is much larger (>10x larger) than any of the Acrs. A fusion of such non-competitive Acrs can be achieved by creating a synthetic construct that will express both Acr open reading frames in frame with one another, separated by a flexible peptide linker. Several lengths and compositions of this linker can be tested to achieve maximal activity. Example VI

Bioinformatics Analysis

BLASTp searches for Aca2 were conducted with WP_019933869.1 from Oceanimonas smirnovii as the query (Pawluk et al., 2016). BLASTp searches for Aca3 were conducted with WP_049360086.1 from Neisseria meningitidis as the query. Information from Makarova et al. (2015), encompassing a representative list of CRISPR-Cas interference proteins, was used to create a sequence similarity dendogram of CRISPR-Cas interference modules for all known CRISPR-Cas types (Makarova et al., 2015). For the phylogenetic analysis of Cas9 protein sequences, a list of 257 representative Cas9 protein sequences was extracted from a previous analysis (Fonfara et al., 2014) and updated with newly deposited sequences in the NCBI Protein database. The list was manually trimmed so that only one representative from each species remained. After alignment of the sequences with MUSCLE (Edgar, 2004), FastTree was used to create an unrooted maximum likelihood tree (Price et al., 2009, 2010). Bootstrap values are shown at each node. Based on the data from Fonfara et al. (2014), each clade was classified into subtype II-A (blue), II-B (yellow), or II-C (purple). Clades on the tree are coloured in red if they belong to any genus where a validated type II-C anti-CRISPR gene or its ortholog was found. Some noteworthy Cas9 proteins are highlighted on the tree by asterisks. Example VII

Plasmid Construction

Acr expression vectors for protein purification DNA sequences encoding candidate anti- CRISPR proteins were synthesized by GenScript (Piscataway, NJ, USA) and subcloned into pHAT4 (Peranen et al., 1996) using NcoI-HindIII restriction sites. The gene encoding AcrE2 was amplified by PCR from Pseudomonas phage JBD88a and ligated into pHAT4 using NcoI- HindIII restriction sites. Table 1 contains the DNA and protein sequences of the anti-CRISPR proteins tested in this study. AcrIIC3Nme was found to be significantly more soluble upon addition of an N-terminal FLAG tag, so that construct was used for in vitro analyses. Example VIII

Cas9:sgRNA Vector For Protein Purification

A DNA encoding a minimal T7 promoter upstream of a random sequence sgRNA (i.e. no genomic target in E. coli) having the following sequence: 5’-TGAGACCAGTCTCGGAAGCTCAAAGGTCTCGTTGTAGCTCCCTTTCTCATTT CGGAA ACGAAATGAGAACCGTTGCTACAATAAGGCCGTCTGAAAAGATGTGC CGCAACGCT CTGCCCCTTAAAGCTTCTGCTTTAAGGGGCATCGTTTATTTCGG TTAAAAAATGCCGT -3’ was synthesized by GenScript (Piscataway, NJ, USA). This insert was cloned into the previously described pMCSG7-NmeCas9 expression vector (Zhang et al., 2015), downstream of the NmeCas9 protein-coding region, into the SalI-XhoI restriction sites. Example IX

Cas9/sgRNA Mammalian Expression Vectors

For editing of DTS3 and DTS7 by both SpyCas9 and NmeCas9 we used Cas9 expression vectors that were identical in all respects [plasmid backbone, CMV IE94 promoter (Villefranc et al., 2007), UTRs, terminal fusions of NLSs and epitope tags, etc.] except for the respective Cas9 ORFs. The SpyCas9 expression plasmid (pEJS24) has been described previously (Bolukbasi et al., 2015) and the NmeCas9 expression plasmid (pEJS424) was generated from (pEJS24) by Cas9 ORF replacement via Gibson assembly (New England Biolabs). Similarly, plasmids for the expression of sgRNAs for each Cas9 ortholog were also identical in all respects except for the sgRNA sequences themselves. The SpyCas9 sgRNA plasmid pLKO.1-puro has been described previously (Kearns et al., 2014), and the NmeCas9 sgRNA expression plasmid (pEJS333) was generated from it by Gibson assembly. The plasmids expressing NmeCas9 and its sgRNA are described in detail elsewhere (Amrani et al., manuscript in preparation). For editing of the N- TS1C, N-TS4B, N-TS4C, N-TS7, N-TS8, N-TS11 and N-TS25 sites, an all-in-one vector (pEJS15) was used expressing both NmeCas9 (under the control of the EF-1a promoter) and its sgRNA (under the control of the U6 promoter). This plasmid, which was derived from pSimpleII (Hou et al., 2013), is also described elsewhere (Amrani et al., manuscript in preparation). The 24-nt guide sequences for each distinct target site were inserted into the sgRNA cassette of pEJS15 by the ligation of synthetic oligonucleotide duplexes into its BsmBI sites. Example X

Acr Vectors For Mammalian Expression

To generate the Acr expression plasmids p427-AcrE2, p430-AcrIIC1Boe, p433- AcrIIC1Nme, p436-AcrIIC2Nme, and p443-AcrIIC3Nme, each ORF was synthesized as a gene block (Integrated DNA Technologies) flanked by XhoI and BstBI sites, with a Kozak consensus sequence upstream of the initiation codon. The synthetic Acr sequences were then inserted into the XhoI and BstBI sites of the pCS2-Dest vector (Addgene). The resulting plasmids placed the Acr-encoding genes under the control of the CMV IE94 promoter. Example XI

Neisseria meningitidis Transformation

Candidate anti-CRISPR genes with the native NmeCas9 promoter and Shine-Dalgarno sequence were cloned into pGCC2, a N. meningitidis vector containing homology arms for integration of the insert into the N. meningitidis chromosome at the nics locus, as described previously (Zhang et al., 2013). The pGCC2 constructs were transformed into N. meningitidis strain 8013, and erythromycin-resistant transformants were selected. Two or three representative transformants per reaction were verified by re-streaking on selective plates twice and then confirmed by PCR on purified genomic DNA. This procedure resulted in N. meningitidis strain 8013 derivatives with chromosomally integrated anti-CRISPR genes under the control of the native promoter of N. meningitidis Cas9. In all cases, the CRISPR locus was sequence-confirmed in the derived strains to ensure that the spacers to be tested for interference activity were intact.

Transformation assays to assess CRISPR-Cas activity of these strains were completed as described previously (Duffin and Seifert, 2012; Zhang et al., 2013), with protospacer 25 (i.e., complementary to the crRNA derived from endogenous CRISPR spacer #25) as the target.

Briefly, 150 ng of plasmids were used per transformation reaction and 10 μL of serial 10-fold dilutions were spotted on GCB plates in triplicate in the presence and absence of appropriate antibiotics.200 μL from the undiluted final transformation mixture were also plated on GCB plates with appropriate antibiotics to enhance detection.

Eight representative transformants per reaction were verified by re-streaking on selective plates twice and then verified by PCRs on cell extracts. Transformation frequencies were reported as antibiotic-resistant cfu/mL divided by total cfu/mL from at least three independent experiments (mean ± s.e.m.). Cloning and purification of anti-CRISPR proteins anti-CRISPR proteins were purified from pHAT4 constructs expressed in E. coli BL21 as described previously (Bondy-Denomy et al., 2015). After elution from Ni-NTA resin, anti-CRISPR proteins were dialyzed in 10 mM Tris pH 7.5, 250mM NaCl, and 5mM ^-mercaptoethanol and incubated with His-tagged Tobacco Etch Virus (TEV) protease overnight at 4°C. A second round of Ni-NTA purification was used to isolate successfully cleaved, untagged anti-CRISPR proteins by collecting the unbound fraction. Example XII

Purification Of Cas9

6xHis-NmeCas9:sgRNA was expressed in E. coli Rosetta (DE3). Cells were grown in Terrific Broth (TB) medium at 37°C to an optical density (OD600 nm) of 0.8 in the Lex

Bubbling System (Structural Genomics Consortium, Toronto, Canada). Protein expression was induced by the addition of 1 mM IPTG for 16 h at 16°C. Cells were lysed by sonication in 50 mM Tris pH 7.5, 500 mM NaCl, 20 mM imidazole, 0.5 mM DTT and 5% glycerol supplemented with 0.5 mM PMSF, lysozyme and protease inhibitor cocktail (Sigma). Clarified lysates were bound in batch to Ni-NTA agarose (Qiagen), and bound protein was eluted with 300 mM imidazole. Purified Cas9:sgRNA was dialysed into 20 mM HEPES pH 7.5, 250 mM NaCl, 5% glycerol, 1 mM DTT and 1 mM PMSF) for protein interaction experiments.6xHis-MBP-tagged AnaCas9 was purified from E. coli BL21 Rosetta cells as described previously (Ma et al., 2015a). Example XIII

Cas9-Anti-CRISPR Protein Pulldown Assays

Untagged anti-CRISPR proteins (after TEV cleavage) were incubated with and without NmeCas9 for 1 hour at 4 °C in binding buffer (20 mM HEPES pH 7.5, 250 mM NaCl, 5% glycerol, 5 mM imidazole), and input fractions were set aside for SDS–PAGE analysis.50 μL 50% slurry Ni-NTA beads were added to each tube. After 30 minutes incubation at 4 °C with rotation, the beads were collected by centrifugation at 3000 rpm for 2 minutes. Beads were washed four times with 1 mL binding buffer supplemented with 20 mM imidazole and collected by centrifugation. Bound proteins were eluted with elution buffer (binding buffer containing 300 mM imidazole). The input and elution fractions were analyzed by SDS-PAGE followed by Coomassie staining. Example XIV

In Vitro DNA Cleavage

NmeCas9 sgRNA derived from spacer (Zhang et al., 2015) was generated by in vitro T7 transcription (Ampliscribe). NmeCas9 (500 nM) was incubated with purified, recombinant anti- CRISPR protein in cleavage buffer [20 mM HEPES-KOH (pH 7.5), 150 mM KCl, 10% glycerol, 1 mM DTT, and 10 mM MgCl ₂ ] for 10 minutes. Next, sgRNA (1:1, 500 nM) was added and the mixture was incubated for another 15 minutes. Linear plasmid containing the target protospacer was added to the Cas9/sgRNA complex at ~5 nM final concentration. The reactions were incubated at 37 °C for 30 minutes and visualized after electrophoresis in a 1% agarose/1xTAE gel. Example XV

Mammalian Genome Editing Plasmids

Plasmids for mammalian expression of NmeCas9, SpyCas9, their respective sgRNAs, and the anti-CRISPR proteins are disclosed herein. Table 4.

Approximately 1.5 x 10 ⁵ mid-passage HEK293T cells cultured at 37°C, 5% CO ₂ in DMEM + 10% FBS + 1% Penicillin/Streptomycin (Gibco) were transiently transfected with 150 ng Cas9- expressing plasmid and 150 ng sgRNA expressing plasmid, using Polyfect transfection reagent (Qiagen) in 24-well plates according to the manufacturer’s instructions. Alternatively, 200 ng of an all-in-one plasmid expressing both NmeCas9 and the appropriate sgRNA was used for transfection.

For experiments that included Acr protein expression, 100 ng of the Acr plasmid was included in the co-transfection mix. 72 hours after transfection, cells were harvested and genomic DNA was extracted with the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer’s instructions.50 ng genomic DNA was used for PCR amplification (High Fidelity 2X PCR Master Mix (New England Biolabs) with primers flanking the targeted site.10 μl of each PCR product was heat denatured, re-annealed, and digested with T7 Endonuclease I (New England Biolabs). The samples were fractionated in a 2.5% agarose/1xTAE gel and quantified with the ImageMaster- TotalLab program. Indel percentages (―% lesion‖ in the figures) were calculated as previously described (Guschin et al., 2010). Example VI

Fluorescence Microscopy Of dNmeCas9

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