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Inhibitors of crispr-cas9 — The Regents Of The University Of California (US20230287058A1)

The Regents Of The University Of California · Google Patents

Google Patents · Patents · License: Open Access

patent, google patents, intellectual property, US20230287058A1, The Regents Of The University Of California, Joseph Bondy-Denomy, en, 2023

ABSTRACT

Abstract

Cas9-inhibiting polypeptide compositions and methods are provided.

Description

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a divisional of U.S. patent application Ser. No. 16/349,253, filed May 10, 2019 (allowed); which is a U.S. National Phase Application Under 371 of PCT/US2017/061932, filed Nov. 16, 2017, which claims benefit of priority to U.S. Provisional Patent Application No. 62/422,850, filed Nov. 16, 2016, which is incorporated by referenced for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant no. OD021344, awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. The Sequence Listing has been filed as an electronic document via EFS-Web in ASCII format encoded as XML. The electronic document, created on Oct. 13, 2022, is entitled â2022-12-08_081906-1350802-224820US_ST26.xmlâ, and is 296,353 bytes in size.

BACKGROUND OF THE INVENTION

The ability to prevent attack from viruses is a hallmark of cellular life. Bacteria employ multiple mechanisms to resist infection by bacterial viruses (phages), including restriction enzymes and CRISPR-Cas systems (Labrie, S. J., Samson, J. E., and Moineau, S. (2010). Nat Rev Micro, 8, 317-327). CRISPR arrays possess the sequence-specific remnants of previous encounters with mobile genetic elements as small spacer sequences located between their clustered regularly interspaced short palindromic repeats (Mojica, F. J. M et al. (2005). J. Mol. Evol., 60, 174-182). These spacers are utilized to generate guide RNAs that facilitate the binding and cleavage of a programmed target (Brouns, S. J. J et al. (2008). Science, 321, 960-964; Garneau, J. E. et al. (2010). Nature, 468, 67-71). CRISPR-associated (cas) genes that are required for immune function are often found adjacent to the CRISPR array (Marraffini, L. A. (2015). CRISPR-Cas immunity in prokaryotes. Nature, 526, 55-61; Wright, A. V., NuÃ±ez, J. K., and Doudna, J. A. (2016). Cell, 164, 29-44). Cas proteins not only carry out the destruction of a foreign genome (Garneau, J. E. et al. (2010). Nature, 468, 67-71), but also facilitate the production of mature CRISPR RNAs (crRNAs) (Deltcheva; Haurwitz, R. E et al. (2010). Science, 329, 1355-1358) and the acquisition of foreign sequences into the CRISPR array (NuÃ±ez, J. K. et al. (2014). Nat. Struct. Mol. Biol, 21, 528-534; Yosef, I., Goren, M. G., and Qimron, U. (2012). Nucleic Acids Research, 40, 5569-5576).

CRISPR-Cas adaptive immune systems are common and diverse in the bacterial world. Six different types (I-VI) have been identified across bacterial genomes (Abudayyeh, O. O et al. (2016). Science aaf5573; Makarova, K. S. et al. (2015). Nat Rev Micro, 13, 722-736). Nat Rev Micro, 13, 722-736), with the ability to cleave target DNA or RNA sequences as specified by the RNA guide. The facile programmability of CRISPR-Cas systems has been widely exploited, opening up the door to many novel genetic technologies (Barrangou, R., and Doudna, J. A. (2016), Nature Biotechnology, 34, 933-941). Most of these technologies use Cas9 from Streptococcus pyogenes (Spy), together with an engineered single guide RNA as the foundation for such applications, including gene editing in animal cells (Cong, L. et al. (2013). Science 339, 819-823; Jinek, M. et al. (2012). Science, 337, 816-821; Mali, P. et al. (2013). Science, 339, 823-826; Qi, L. S. et al. (2013). Cell, 152, 1173-1183). Additionally, Cas9 orthologs within the II-A subtype have been investigated for gene editing applications (Ran, F. A. et al. (2015). Nature 520, 186-191), and new Class 2 CRISPR single protein effectors such as Cpf1 (Type V (Zetsche, B. et al. (2015). Cell, 163, 759-771)) and C2c2 (Type VI (Abudayyeh, O. O et al. (2016). Science aaf5573; East-Seletsky, A. et al. (2016). Nature 538, 270-273) are being characterized. Class 1 CRISPR-Cas systems (Type I, III, and IV) are RNA-guided multi-protein complexes and thus have been overlooked for most genomic applications due to their complexity. These systems are, however, the most common in nature being found in nearly half of all bacteria and Ë85% of archaea (Makarova, K. S. et al. (2015). Nat Rev Micro, 13, 722-736). Nat Rev Micro, 13, 722-736).

In response to the bacterial war on phage infection, phages, in turn, often encode inhibitors of bacterial immune systems that enhance their ability to lyse their host bacterium or integrate into its genome (Samson, J. E. et al. (2013). Nat Rev Micro, 11, 675-687). The first examples of phage-encoded âanti-CRISPRâ proteins came for the (Class 1) type I-E and I-F systems in Pseudomonas aeruginosa (Bondy-Denomy et al. (2013). Nature, 493, 429-432; Pawluk, A. et al. (2014). mBio 5, e00896). Remarkably, ten type I-F anti-CRISPR and four type I-E anti-CRISPR genes have been discovered to date (Pawluk, A. et al. (2016). Nature Microbiology, 1, 1-6), all of which encode distinct, small proteins (50-150 amino acids), previously of unknown function. Our biochemical investigation of four I-F anti-CRISPR proteins revealed that they directly interact with different Cas proteins in the multi-protein CRISPR-Cas complex to prevent either the recognition or cleavage of target DNA (Bondy-Denomy, J et al. (2015). Nature, 526, 136-139). Each protein has a distinct sequence, structure, and mode of action (Maxwell, K. L. et al. (2016). Nature Communications, 7, 13134; Wang, X. (2016). Nat. Struct. Mol. Biol 23, 868-870). These findings support the independent evolution of CRISPR-Cas inhibitors and suggests that many more are yet to be discovered. In this light, a recent paper utilized the conservation of signature anti-CRISPR associated (aca) gene with a predicted helix-turn-helix (HTH) motif to identify anti-CRISPR genes outside of P. aeruginosa . This led to the authors finding anti-CRISPRs across proteobacteria, broadly spanning the type I-F CRISPR-Cas phylogeny (Pawluk, A. et al. (2016). Nature Microbiology, 1, 1-6). This suggests that anti-CRISPRs may exist for all CRISPR systems, with methods needed to enable their discovery.

The type I anti-CRISPRs in P. aeruginosa are expressed from integrated phage genomes (prophages), leading to the constitutive inactivation of the host CRISPR-Cas system (Bondy-Denomy et al. (2013). Nature, 493, 429-432. This can often lead to a situation where a prophage possesses a DNA target with perfect identity to a co-occurring CRISPR spacer in the same cell, called âself-targetingâ ( FIG. 1 A ). This situation makes CRISPR-Cas inactivation a requirement for survival, as in the absence of prophage anti-CRISPR genes, the host genome is cleaved in the act of targeting the prophage (Bondy-Denomy et al. (2013). Nature, 493, 429-432; Edgar, R., and Qimron, U. (2010). J. Bacteriol 192, 6291-6294). Expression of an anti-CRISPR neutralizes this risk, however, allowing lysogen survival. We surmised that genomes possessing a CRISPR system with apparent self-targeting would be candidates for the identification of new CRISPR-Cas inhibitors. Here, we describe the identification of previously unknown phage-encoded CRISPR-Cas9 inhibitors in Listeria monocytogenes using a bioinformatics approach to identify incidents of self-targeting. We show that two of these inhibitors can also block the activity of S. pyogenes Cas9 in bacterial and human cells.

Definitions

The term ânucleic acidâ or âpolynucleotideâ refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The term âgeneâ means the segment of DNA involved in producing a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

A âpromoterâ is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. The promoter can be a heterologous promoter.

An âexpression cassetteâ is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter. The promoter can be a heterologous promoter. In the context of promoters operably linked to a polynucleotide, a âheterologous promoterâ refers to a promoter that would not be so operably linked to the same polynucleotide as found in a product of nature (e.g., in a wild-type organism).

âPolypeptide,â âpeptide,â and âproteinâ are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

âConservatively modified variantsâ applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, âconservatively modified variantsâ refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are âsilent variations,â which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a âconservatively modified variantâ where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. In some cases, conservatively modified variants of Cas9 or sgRNA can have an increased stability, assembly, or activity as described herein.

The following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins, W. H. Freeman and Co., N. Y. (1984)).

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

In the present application, amino acid residues are numbered according to their relative positions from the left most residue, which is numbered 1, in an unmodified wild-type polypeptide sequence.

As used in herein, the terms âidenticalâ or percent âidentity,â in the context of describing two or more polynucleotide or amino acid sequences, refer to two or more sequences or specified subsequences that are the same. Two sequences that are âsubstantially identicalâ have at least 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection where a specific region is not designated. With regard to polynucleotide sequences, this definition also refers to the complement of a test sequence. With regard to amino acid sequences, in some cases, the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST 2.0 algorithm and the default parameters discussed below are used.

A âcomparison windowâ, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

<div id="p-0022" num="0030" class="descri

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant no. OD021344, awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

BACKGROUND OF THE INVENTION

Definitions

The following eight groups each contain amino acids that are conservative substitutions for one another:

In the present application, amino acid residues are numbered according to their relative positions from the left most residue, which is numbered 1, in an unmodified wild-type polypeptide sequence.

An algorithm for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithm, which are described in Altschul et al., (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information website, ncbi.nlm.nih.gov. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=â2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The âCRISPR/Casâ system refers to a class of bacterial systems for defense against foreign nucleic acid. CRISPR/Cas systems are found in a wide range of eubacterial and archaeal organisms. CRISPR/Cas systems include type I, II, III, V, and VI sub-types. Wild-type type II CRISPR/Cas systems utilize the RNA-mediated nuclease, Cas9 in complex with guide and activating RNA to recognize and cleave foreign nucleic acid.

Cas9 homologs are found in a wide variety of eubacteria, including, but not limited to bacteria of the following taxonomic groups: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chlroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 polypeptide is the Streptococcus pyogenes Cas9 polypeptide. Additional Cas9 proteins and homologs thereof are described in, e.g., Chylinksi, et al., RNA Biol. 2013 May 1; 10(5): 726-737; Nat. Rev. Microbiol. 2011 June; 9(6): 467-477; Hou, et al., Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15644-9; Sampson et al., Nature. 2013 May 9; 497(7448):254-7; and Jinek, et al., Science. 2012 Aug. 17; 337(6096):816-21. The Cas9 protein can be nuclease defective. For example, the Cas9 protein can be a nicking endonuclease that nicks target DNA, but does not cause double strand breakage. Cas9 can also have both nuclease domains deactivated to generate âdead Cas9â (dCas9), a programmable DNA-binding protein with no nuclease activity. In some embodiments, dCas9 DNA-binding is inhibited by the polypeptides described herein.

BRIEF SUMMARY OF THE INVENTION

In one aspect, methods of inhibiting a Cas9 polypeptide in a cell are provided. In some embodiments, the method comprises,

introducing a Cas9-inhibiting polypeptide into a cell, wherein: the Cas9-inhibiting polypeptide is heterologous to the cell, and the Cas9-inhibiting polypeptide is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%) identical to any one or more of SEQ ID NO: 1-170, thereby inhibiting the Cas9 polypeptide in a cell. In some embodiments, the method comprises contacting the Cas9 inhibiting polypeptide with a Cas9 polypeptide in the cell. In some embodiments, the method comprises contacting the Cas9 inhibiting polypeptide with other components of the CRISPR-Cas9 system in the cell, thereby indirectly inhibiting Cas9 polypeptide activity.

In some embodiments, the Cas9-inhibiting polypeptide comprises one of SEQ ID NO: 1-170.

In some embodiments, the cell comprises the Cas9 polypeptide before the introducing. In some embodiments, the cell comprises an expression cassette comprising a promoter operably linked to a polynucleotide encoding the Cas9 polypeptide. In some embodiments, the promoter is inducible and the method comprises contacting the cell with an agent or condition that induces expression of the Cas9 polypeptide in the cell prior to the introducing.

In some embodiments, the cell comprises the Cas9 polypeptide after the introducing. In some embodiments, the promoter is inducible and the method comprises contacting the cell with an agent or condition that induces expression of the Cas9 polypeptide in the cell after to the introducing.

In some embodiments, the introducing comprises expressing the Cas9-inhibiting polypeptide in the cell from an expression cassette that is present in the cell and heterologous to the cell, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding the Cas9-inhibiting polypeptide. In some embodiments, the promoter is an inducible promoter and the introducing comprises contacting the cell with an agent that induces expression of the Cas9-inhibiting polypeptide.

In some embodiments, the introducing comprises introducing an RNA encoding the Cas9-inhibiting polypeptide into the cell and expressing the Cas9-inhibiting polypeptide in the cell from the RNA.

In some embodiments, the introducing comprises inserting the Cas9-inhibiting polypeptide into the cell or contacting the cell with the Cas9-inhibiting polypeptide.

In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a blood or an induced pluripotent stem cell. In some embodiments, the cell is a prokaryotic cell.

In some embodiments, the method occurs ex vivo. In some embodiments, the cells are introduced into a mammal after the introducing and contacting. In some embodiments, the cells are autologous to the mammal.

Also provided is a cell (optionally isolated) comprising a Cas9-inhibiting polypeptide, wherein the Cas9-inhibiting polypeptide is heterologous to the cell and the Cas9-inhibiting polypeptide is substantially identical to any one or more of SEQ ID NO: 1-170. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell In some embodiments, the cell is a prokaryotic cell.

Also provided is a polynucleotide comprising a nucleic acid encoding a Cas9-inhibiting polypeptide. In some embodiments, the Cas9-inhibiting polypeptide is substantially identical to any one or more of SEQ ID NO: 1-170. In some embodiments, the polynucleotide comprises an expression cassette, the expression cassette comprising a promoter operably linked to the nucleic acid. In some embodiments, the promoter is heterologous to the polynucleotide encoding the Cas9-inhibiting polypeptide. In some embodiments, the promoter is inducible. In some embodiments, the polynucleotide is DNA or RNA.

Also provided is a vector comprising the expression cassette as described above or elsewhere herein. In some embodiments, the vector is a viral vector.

Also provided is a (optionally isolated) Cas9-inhibiting polypeptide. In some embodiments, the Cas9-inhibiting polypeptide is substantially identical to any one or more of SEQ ID NO:1-170.

Also provided is a pharmaceutical composition comprising the polynucleotide or the polypeptide as described above or elsewhere herein.

Also provided is a delivery vehicle comprising the polynucleotide or the polypeptide as described above or elsewhere herein. In some embodiments, the delivery vehicle is a liposome or nanoparticle.

Other aspects are described in the remainder of this document.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A-C . A survey for CRISPR-Cas9 genomic self-targeting (ST) in Listeria monocytogenes.

FIG. 1 A A schematic depicting the principle of genomic self-targeting, where a mobile genetic element (MGE) possesses a target sequence for a spacer in a CRISPR array in the same genome. CRISPR-Cas9 function in this âself-targeting genomeâ is presumably inactive for continued cell viability.

FIG. 1 B The abundance of genomes with cas9-linked self-targeting (red) and those without ST (grey), in L. monocytogenes genomes.

FIG. 1 C An example of an ST event, where spacer 16 in the CRISPR array of strain J0161 has a perfect PAM and protospacer match with a resident prophage (ÏJ0161 (spacer sequences: crRNA (SEQ ID NO:171), protospacer and protospacer complement (SEQ ID NOS:172-173)).

FIG. 2 A-D . A prophage from L. monocytogenes J0161 contains two CRISPR-Cas9 inhibitor genes.

FIG. 2 A The type II-A CRISPR-Cas locus in L. monocytogenes 10403s. Four cas genes are indicated, along with tracrRNA and CRISPR array, containing 30 spacers. The predicted direction of transcription is indicated with black arrows. Subsequent experiments utilize a non-targeted plasmid (pNT) and a targeted plasmid (pT) that has a protospacer matching spacer 1 in this strain.

FIG. 2 B Representative pictures of transformed colonies after CRISPR-Cas-targeted (pT) or non-targeted (pNT) plasmids were electroporated into phage-cured (Ïcure) strains of L. monocytogenes 10403s and wild type (wt) J0161 (contains the ÏJ0161a prophage) is shown in red to denote self-targeting. Analyzed Ïcure 10403s variants include a cas9-deletion strain (Îcas9), a lysogen of ÏJ0161a (::ÏJ0161a), strains constitutively expressing individual CRISPR-Cas9 inhibitor genes from ÏJ0161a (+acrIIA1, +acrIIA2) and a lysogen of ÏJ0161a with CRISPR-Cas9 inhibitor genes deleted (:: ÏJ0161aÎacrIIA1-2). See FIG. 7 for a comparison of wt and Ïcure 10403s.

FIG. 2

C 10403s Ïcure and wt J0161 strains were assessed for transformation efficiency. The 10403s Ïcure cas9-deletion strain (Îcas9), constitutively expressed cas9 (Îcas9+cas9) and ÏJ0161a lysogens of these strains (::ÏJ0161a) were analyzed. Error bars reflect the standard deviation of three biological replicates. L.D. limit of detection.

FIG. 2 D Comparison of the open reading frames from two similar prophages from L. monocytogenes 10403s and J0161. Unique genes (red) comprising ten fragments of ÏJ0161 were tested for CRISPR-Cas9 inhibition in 10403s. n.e., No effect on CRISPR-Cas9 activity, tox., fragment toxic when expressed, t., location of self-targeted protospacer. The encircled fragment exhibited anti-CRISPR activity with two genes (acrAII1, acrAII2) independently capable of inhibiting CRISPR-Cas activity. Conserved (grey) genes were not tested. For reference, phage genes involved in cell lysis, capsid assembly and host integration (int.) are labeled.

FIG. 3 A-C . Genomic organization and prevalence of acrIIA genes

FIG. 3 A The genomic context of acrIIA1 (1) and its homolog from L. monocytogenes (orfD) are depicted to scale as cartoons with acrIIA1 homologs in vertical alignment. Typically, acrIIA genes are encoded within prophages adjacent to or near the phage lysin (ply) gene. Genomic neighbors of acrIIA1 and orfD (acrIIA1-4, orfA-E) are shown. Individual genes (***) were assayed for CRISPR-Cas9 inhibition in L. monocytogenes 10403s (see FIG. 9 ). Helix-turn-helix (HTH) and AP2 DNA binding motifs were detected in some proteins using hidden markov model (HMM) prediction software (SÃ¶ding, J., Biegert, A., and Lupas, A. N. (2005). Nucleic Acids Research, 33, W244-W248).

FIG. 3 B Pie-graph representation of the frequency of each acrIIA gene co-occurrences

FIG. 3 C Pie-graph representation of the prevalence of acrIIA and cas9 genes in the L. monocytogenes pan-genome. See Supplementary Table 1 for relevant accession numbers.

FIG. 4 A . Phylogenetic analysis of AcrIIA1-4 homologs

A phylogenetic reconstruction of full-length protein sequences identified following an iterative psi-BLASTp search to query all non-redundant protein sequences within GenBank for ( FIG. 4 A ) AcrIIA1.

BLASTp was used to construct a similar tree for ( FIG. 4 B ) AcrIIA2, ( FIG. 4 C ) AcrIIA3, and FIG. 4 D AcrIIA4 (see Methods). Selected bootstrapping support values are denoted with filled ovals (â¥90%), open rectangles (â¥70%) or dashed lines (<70%). The sequence family that is boxed-in represents the family that was tested for anti-CRISPR function. Other homologs reflect distinct sequence families present in the genomes described under the tree.

FIG. 5 A-D . Inhibition of Streptococcus pyogenes dCas9 and Cas9.

FIG. 5 A A schematic outlining the experimental setup, where single-cell fluorescence of E. coli BW25113 with Streptococcus pyogenes (Spy) dCas9 and a guide RNA targeted towards a chromosomal red fluorescent protein (RFP) gene was measured.

FIG. 5 B Candidate (orf) and validated (acr) acrIIA genes were tested for their ability to inhibit dCas9-based repression. Measurements taken reflect the median RFP fluorescence value of a single cell in a unimodal population normalized for each candidate gene to a guide RNA-free control. Error bars represent the standard deviation of at least three biological replicates. See FIG. 2 and FIG. 9 for gene-identification information.

FIG. 5 C A schematic outlining the experimental setup, where HEK293T cells with a doxycycline-inducible eGFP cassette were transfected with a plasmid encoding a single transcript tracrRNA/eGFP-targeting guide RNA and NLS-SpyCas9 alongside expression constructs encoding one of five codon-optimized phage genes at different ratios. The percent of eGFP positive cells was measured 12 hours after induction by flow cytometry.

FIG. 5 D Average percent of eGFP positive cells is depicted+/âstandard deviation across biological triplicates. An increasing amount of inhibitor plasmid (in ng) was added from left to right, at a ratio to the Cas9/sgRNA plasmid of 1:1 and 3:1. Data were normalized to transfection with no phage ORF as the baseline.

FIG. 6 is a cartoon depiction of AcrIIA-inhibition of Cas9.

FIG. 7 A-F . Self-Targeting by CRISPR-Cas9 in Listeria monocytogenes J0161 is Not Associated with Loss-of-Function Mutations, Related to FIG. 1

FIG. 7 A Comparison of type II-A CRISPR-cas loci from Streptococcus pyogenes SF370 (Spy_SF370), Listeria monocytogenes 10403s (Lmo_10403s), Listeria monocytogenes J0161 (Lmo_J0161) and Listeria innocua (Lin_Clip11262). Percent identity between Cas9 protein sequences is shown.

FIG. 7 B The CRISPR array of self-targeting strain Lmo J0161. A type II-A CRISPR array, predicted by the CRISPRDetect web utility is shown (Spacer Sequence: SEQ ID NOS:174-192, respectively; Repeat Sequence: SEQ ID NO:193). The self-targeting spacer (number 16 (SEQ ID NO:189) is boxed. In bold, are the RNA-coding nucleotides responsible for target recognition.

FIG. 7 C A modified CRISPRtarget output, depicting self-targeting by L. monocytogenes J0161. The predicted crRNA processing site is identified by the wedge icon. (sequences: crRNA (SEQ ID NO:194), target DNA and target DNA complement (SEQ ID NOS:195-196)).

FIG. 7 D Alignment of tracrRNA loci. (Spy_SF370 (SEQ ID NO:197); Lmo_10403s (SEQ ID NO:198); Lmo_J0161 (SEQ ID NO:199): Lin_Clip11262 (SEQ ID NO:200).

FIG. 7 E Alignment of CRISPR loci. (Spy_SF370 (SEQ ID NO:201); Lmo_10403s (SEQ ID NO:202); Lmo_J0161 (SEQ ID NO:203): Lin_Clip11262 (SEQ ID NO:204).

FIG. 7 F Alignment of Cas9 protein sequences. Residues with essential chemical functionalities are boxed. (Spy_SF370 (SEQ ID NO:205); Lmo_10403s (SEQ ID NO:206); Lmo_J0161 (SEQ ID NO:207): Lin_Clip11262 (SEQ ID NO:208).

FIG. 8 . The Ï10403s Prophage Does Not Influence Plasmid Targeting in L. monocytogenes 10403s, Related to FIG. 2 A-D . Representative plates depicting colonies after transformation and selection for targeted (pT; pRAU31) or non-targeted (pNT; pRAU29) plasmids. Wild type (wt) and nonlysogenic (Ïcure) strains of 10403s were analyzed. See Table S1 for additional information pertinent to plasmid and strain design and nomenclature.

FIG. 9 . Fragments of the ÏJ0161a Prophage that were Screened for CRISPR-Cas9 Inhibition Activity in L. monocytogenes 10403s, Related to FIG. 2 . Representative plates depicting colonies after transformation and selection for targeted (pT; pRAU31) or non-targeted (pNT; pRAU29) plasmids. DNA sequence information is provided for all phage fragments as they are named in FIG. 2 d . See Table 51 for additional information pertinent to plasmid and strain design and nomenclature.

FIG. 10 A-B . Individual Genes that were Screened for CRISPR-Cas9 Inhibition Activity in L. monocytogenes 10403s, Related to FIG. 2 B and FIG. 3 A . Representative plates depicting colonies after transformation and selection for targeted (pT; pRAU31) or non-targeted (pNT; pRAU29) plasmids. Given names, locus tags, accession numbers and DNA sequence information is provided for all candidate type II-A CRISPR-Cas inhibitors. See Table S1 for additional information pertinent to plasmid and strain design and nomenclature.

FIG. 11 A-B . Toxicity of an AcrIIA3 Homolog from S. pyogenes in E. coli , Related to FIG. 5 .

FIG. 11 A Distribution of single-cell RFP fluorescence values for E. coli CRISPRi reporter strains with and without expression of AcrIIA proteins. Expression of AcrIIA proteins leads to unimodal shift in population fluorescence towards the sgRNA (no CRISPRi knockdown) state, indicating a uniform disruption of CRISPRi activity. Strains were grown for 2.5 hr in the presence of IPTG to induce CRISPRi, with or without expression of the AcrIIA inhibitor.

FIG. 11 B Expression of Spy AcrIIA3 is toxic in E. coli . In the presence of IPTG (CRISPRi induction) and arabinose (AcrIIA3 induction), Spy AcrIIA3 is toxic in the presence or absence of sgRNA, indicating that its toxicity is independent of CRISPRi activity.

FIG. 12 . Vector Map file for pPL2oexL, Related to FIG. 2 and FIG. 3 . Genes and phage fragments to be tested for CRISPR-Cas9 inhibition in L. monocytogenes 10403s were cloned into pPL2oexL between pHyper and the FLAG tag. Native stop codons were included in pPL2oexL derivatives.

FIG. 13 : acrIIA1 is very widespread across Firmicutes homologs are likely to inhibit Cas9 function in the organisms in which they are found. To identify new homologs of acrIIA genes with anti-CRISPR function, distinct members from the phylogenetic trees shown here were tested for anti-CRISPR activity in cell based assays. These homologs of known anti-CRISPR genes are being tested in a foreign bacterial system ( Pseudomonas aeruginosa ) to identify those with direct activity against SpyCas9.

FIG. 14 : Phage plaque assays showing ten-fold dilutions of a control phage (D3) or a phage targeted (JBD30) by SpyCas9 with a JBD30-specific sgRNA in a heterologous host ( Pseudomonas aeruginosa ). Expression of the indicated anti-CRISPR acrIIA4 (positive control) or acrIIA1 inactivates SpyCas9.

FIG. 15 A-B : 15 A) A multi-sequence alignment of acrIIA2 homologs found in different Listeria mobile elements. (AcrIIA2a.1 (SEQ ID NO:69); AcrIIA2a.2 (SEQ ID NO:84); AcrIIA2b.1 (SEQ ID NO:108); AcrIIA2b.3 (SEQ ID NO:209); AcrIIA2c.1 (SEQ ID NO:97); and AcrIIA2c.2 (SEQ ID NO:98)). Tested homologs are indicated with colored arrows and a summary of the results are shown below the alignment, 15 B) A table summarizing the sequence identity (at the amino acid level) between the different homologs and their accession numbers.

FIG. 16 : Bacteriophage plaque assays with ten-fold serial dilutions phage JBD30 spotted on top of a lawn of P. aeruginosa expressing SpyCas9 and a sgRNA targeting phage JBD30. Phages will plaque in the absence of CRISPR activity. Cas9 and the sgRNA are induced with increasing amounts of arabinose from left to right (0.001%, 0.01%, 0.1%). In the absence of Cas9 (Îcas9), the phages plaque fully, but in the present of Cas9 but no anti-CRISPR (vector), plaguing is reduced as Cas9 is induced. The provision of acrIIA4 (positive control) fully blocks Cas9 at all levels. Only acrIIA2b.3 is comparable to acrIIA4 for its activity. The original acrIIA2a.1 is only partially active, with a slight improvement seen with acrIIA2b.1.

FIG. 17 : Homologs of acrIIA3b found in Streptococcus species were tested for anti-CRISPR activity in heterologous system ( P. aeruginosa ) expressing SpyCas9 and an sgRNA. A summary of the results (data in FIG. 6 ) are shown in the table, indicating the species of origin for the anti-CRISPR, the sequence identity of Cas9 in that species to S. pyogenes , and similar information for the anti-CRISPR. Given the previously observed toxicity of acrIIA3a and acrIIA3b, these new proteins were assessed for toxicity (toxic?) and anti-CRISPR function (acr?).

FIG. 18 : Spot titration of bacterial cells on LB agar plates. SpyCas9 was programmed with an sgRNA targeting the P. aeruginosa genome, thus killing the cell. Cells only survive if the anti-CRISPR is functional. Plates are showing 10-fold serial dilutions of cells plated on non-inducing (left column), ACR inducing only (middle column, to test ACR toxicity), or Cas9/sgRNA/ACR inducing plates (right column, to test ACR function). Genome being cleaved by Cas9 leads to death, unless anti-CRISPR blocks Cas9 function. Colonies on right-most panels indicate ACR activity.

FIG. 19 : Summary of some of the data from Example 2.

DETAILED DESCRIPTION OF THE INVENTION

Several polypeptide inhibitors (âCas9-inhibiting polypeptidesâ) of Cas9 nuclease have been identified from phage. The Cas9-inhibiting polypeptides initially discovered from phage were designated AcrIIA1, AcrIIA2, AcrIIA3, and AcrIIA4.

The Cas9-inhibiting polypeptides described herein can be used in many aspects to inhibit unwanted Cas9 activity. For example, one or more Cas9-inhibiting polypeptide can be used to regulate Cas9 in genome editing, thereby allowing for some Cas9 activity prior to introduction of the Cas9-inhibiting polypeptide. This can be helpful, for example, in limiting off-target effects of Cas9. This and other uses are described in more detail below.

As set forth in the examples and sequence listing, a large number of Cas9-inhibiting polypeptides have been discovered. Examples of exemplary Cas9-inhibiting polypeptides include proteins comprising any of SEQ ID NOs: 1-169, or substantially (e.g., at least 50, 60, 70, 75, 80, 85, 90, 95, or 98%) identical amino acid sequences. In some embodiments, the polypeptides, in addition to having one of the above-listed sequences, will include other amino acid sequences or other chemical moieties (e.g., detectable labels) at the amino terminus, carboxyl terminus, or both. Additional amino acid sequences can include, but are not limited to tags, detectable markers, or nuclear localization signal sequences.

As noted in the examples, a number of the Cas9-inhibiting polypeptides have been shown to inhibit L. monocytogenes Cas9 as well as S. pyogenes (Spy) Cas9. It is believed and expected that the Cas9-inhibiting polypeptides described herein will also similarly inhibit other block II-A Cas9 proteins. As used herein, a âCas9-inhibiting polypeptideâ is a protein that inhibits function of the Cas9 enzyme in L. monocytogenes during a transformation efficiency assay. When a plasmid bearing a targeted DNA sequence and protospacer adjacent motif (PAM) is used to transform a strain with intact Cas9 function, the transformation event is prevented by Cas9, generating miniscule colonies under selection. This is compared to a plasmid with a non-targeted DNA sequence, which produces normal sized colonies when used to transform L. monocytogenes . The expression of a Cas9 inhibitor neutralizes Cas9 activity and leads to transformed, normal sized colonies of both the targeted and non-targeted plasmid. While it is believed the Cas9-inhibiting polypeptides' inhibitory activity can be measured in other ways, the above assay, presented in more detail in the Examples, is the assay for determining whether the Cas9-inhibiting polypeptide have activity.

The Cas9-inhibiting polypeptides can be introduced into any cell to inhibit Cas9 in that cell. In some embodiments, the cell contains Cas9 protein when the Cas9-inhibiting polypeptide is introduced into the cell. In other embodiments, the Cas9-inhibiting polypeptide is introduced into the cell and then Cas9 polypeptide is introduced into the cell.

Introduction of the Cas9-inhibiting polypeptides into the cell can take different forms. For example, in some embodiments, the Cas9-inhibiting polypeptides themselves are introduced into the cells. Any method for introduction of polypeptides into cells can be used. For example, in some embodiments, electroporation, or liposomal or nanoparticle delivery to the cells can be employed. In other embodiments, a polynucleotide encoding a Cas9-inhibiting polypeptide is introduced into the cell and the Cas9-inhibiting polypeptide is subsequently expressed in the cell. In some embodiments, the polynucleotide is an RNA. In some embodiments, the polynucleotide is a DNA.

In some embodiments, the Cas9-inhibiting polypeptide is expressed in the cell from RNA encoded by an expression cassette, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding the Cas9-inhibiting polypeptide. In some embodiments, the promoter is heterologous to the polynucleotide encoding the Cas9-inhibiting polypeptide. Selection of the promoter will depend on the cell in which it is to be expressed and the desired expression pattern. In some embodiments, promoters are inducible or repressible, such that expression of a nucleic acid operably linked to the promoter can be expressed under selected conditions. In some examples, a promoter is an inducible promoter, such that expression of a nucleic acid operably linked to the promoter is activated or increased.

An inducible promoter may be activated by presence or absence of a particular molecule, for example, doxycycline, tetracycline, metal ions, alcohol, or steroid compounds. In some embodiments, an inducible promoter is a promoter that is activated by environmental conditions, for example, light or temperature. In further examples, the promoter is a repressible promoter such that expression of a nucleic acid operably linked to the promoter can be reduced to low or undetectable levels, or eliminated. A repressible promoter may be repressed by direct binding of a repressor molecule (such as binding of the trp repressor to the trp operator in the presence of tryptophan). In a particular example, a repressible promoter is a tetracycline repressible promoter. In other examples, a repressible promoter is a promoter that is repressible by environmental conditions, such as hypoxia or exposure to metal ions.

In some embodiments, the polynucleotide encoding the Cas9-inhibiting polypeptide (e.g., as part of an expression cassette) is delivered to the cell by a vector. For example, in some embodiments, the vector is a viral vector. Exemplary viral vectors can include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, and lentiviral vectors.

In s

CLAIMS

Claims ( 27 )

1 . A method of inhibiting a Cas9 polypeptide in a cell, the method comprising,

introducing a Cas9-inhibiting polypeptide into a cell, wherein:

the Cas9-inhibiting polypeptide is heterologous to the cell, and

the Cas9-inhibiting polypeptide is substantially (e.g., at least 60%, 70%, 80%, 90%, 95%) identical to any one or more of SEQ ID NO: 1-170;

thereby inhibiting the Cas9 polypeptide in a cell.

2 . The method of claim 1 , comprising contacting the Cas9 inhibiting polypeptide with a Cas9 polypeptide in the cell.

3 . The method of claim 1 , wherein the Cas9-inhibiting polypeptide comprises one of SEQ ID NO: 1-170.

4 . The method of claim 1 , wherein the cell comprises the Cas9 polypeptide before the introducing.

5 . The method of claim 4 , wherein the cell comprises an expression cassette comprising a promoter operably linked to a polynucleotide encoding the Cas9 polypeptide.

6 . The method of claim 5 , wherein the promoter is inducible and the method comprises contacting the cell with an agent or condition that induces expression of the Cas9 polypeptide in the cell prior to the introducing.

7 . The method of claim 1 , wherein the cell comprises the Cas9 polypeptide after the introducing.

8 . The method of claim 7 , wherein the promoter is inducible and the method comprises contacting the cell with an agent or condition that induces expression of the Cas9 polypeptide in the cell after to the introducing.

9 . The method of claim 1 , wherein the introducing comprises expressing the Cas9-inhibiting polypeptide in the cell from an expression cassette that is present in the cell and heterologous to the cell, wherein the expression cassette comprises a promoter operably linked to a polynucleotide encoding the Cas9-inhibiting polypeptide.

10 . The method of claim 9 , wherein the promoter is an inducible promoter and the introducing comprises contacting the cell with an agent that induces expression of the Cas9-inhibiting polypeptide.

11 . The method of claim 1 , wherein the introducing comprises introducing an RNA encoding the Cas9-inhibiting polypeptide into the cell and expressing the Cas9-inhibiting polypeptide in the cell from the RNA.

12 . The method of claim 1 , wherein the introducing comprises inserting the Cas9-inhibiting polypeptide into the cell or contacting the cell with the Cas9-inhibiting polypeptide.

13 - 16 . (canceled)

17 . The method of claim 1 , wherein the method occurs ex vivo.

18 . The method of claim 17 , wherein the cells are introduced into a mammal after the introducing and contacting.

19 . (canceled)

20 . (canceled)

21 . A cell comprising a Cas9-inhibiting polypeptide, wherein the Cas9-inhibiting polypeptide is heterologous to the cell and the Cas9-inhibiting polypeptide is substantially identical to any one or more of SEQ ID NO: 1-170.

22 - 25 . (canceled)

26 . A polynucleotide comprising a nucleic acid encoding the Cas9-inhibiting polypeptide of claim 33 .

27 - 30 . (canceled)

31 . A vector comprising the polynucleotide of claim 26 .

32 . (canceled)

33 . An isolated Cas9-inhibiting polypeptide, wherein the Cas9-inhibiting polypeptide is substantially identical to any one or more of SEQ ID NO:1-170.

34 . A pharmaceutical composition comprising the polypeptide of any of claim 33 .

35 . A delivery vehicle comprising the polypeptide of claim 33 .

36 . (canceled)

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