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• W2987201345 abstract "•Anti-CRISPR AcrIIA5 potently inhibits all Cas9 homologs used in genome editing•AcrIIA5 functions well in a variety of mammalian cell genome-editing applications•The AcrIIA5 functional mechanism leads to sgRNA cleavage CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory activity is generally restricted to specific Cas9 homologs. Since Cas9 homologs vary in important properties, differing Cas9s may be optimal for particular genome-editing applications. To facilitate the practical exploitation of multiple Cas9 homologs, here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing. We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction. CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory activity is generally restricted to specific Cas9 homologs. Since Cas9 homologs vary in important properties, differing Cas9s may be optimal for particular genome-editing applications. To facilitate the practical exploitation of multiple Cas9 homologs, here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing. We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction. CRISPR-Cas9 systems combine a single effector protein, Cas9, with a single-guide RNA (sgRNA) molecule to target specific DNA sequences for precise genome manipulation. Their ability to program these systems to target any desired DNA sequence has led to their widespread usage for creating genomic knockouts and knockins, editing single bases, and gene activation and silencing (Doudna and Charpentier, 2014Doudna J.A. Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9.Science. 2014; 346: 1258096Crossref PubMed Scopus (3615) Google Scholar, Hess et al., 2017Hess G.T. Tycko J. Yao D. Bassik M.C. Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes.Mol. Cell. 2017; 68: 26-43Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, Komor et al., 2017Komor A.C. Badran A.H. Liu D.R. CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes.Cell. 2017; 168: 20-36Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar). However, there are concerns about the ability to safely and effectively control this technology, particularly in the case of applications like gene drives (Baltimore et al., 2015Baltimore D. Berg P. Botchan M. Carroll D. Charo R.A. Church G. Corn J.E. Daley G.Q. Doudna J.A. Fenner M. et al.Biotechnology. A prudent path forward for genomic engineering and germline gene modification.Science. 2015; 348: 36-38Crossref PubMed Scopus (432) Google Scholar, Gantz and Bier, 2015Gantz V.M. Bier E. Genome editing. The mutagenic chain reaction: a method for converting heterozygous to homozygous mutations.Science. 2015; 348: 442-444Crossref PubMed Scopus (368) Google Scholar, Hammond et al., 2016Hammond A. Galizi R. Kyrou K. Simoni A. Siniscalchi C. Katsanos D. Gribble M. Baker D. Marois E. Russell S. et al.A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.Nat. Biotechnol. 2016; 34: 78-83Crossref PubMed Scopus (677) Google Scholar). One mechanism by which CRISPR-Cas9 activity can be controlled is through the use of small, naturally occurring protein inhibitors known as anti-CRISPRs (Borges et al., 2017Borges A.L. Davidson A.R. Bondy-Denomy J. The Discovery, Mechanisms, and Evolutionary Impact of Anti-CRISPRs.Annu. Rev. Virol. 2017; 4: 37-59Crossref PubMed Scopus (114) Google Scholar, Pawluk et al., 2018Pawluk A. Davidson A.R. Maxwell K.L. Anti-CRISPR: discovery, mechanism and function.Nat. Rev. Microbiol. 2018; 16: 12-17Crossref PubMed Scopus (201) Google Scholar). These proteins have been shown to function as off switches for CRISPR-Cas9 genome editing in human cells (Lee et al., 2018Lee J. Mir A. Edraki A. Garcia B. Amrani N. Lou H.E. Gainetdinov I. Pawluk A. Ibraheim R. Gao X.D. et al.Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and AcrIIC5 Anti-CRISPR Proteins.MBio. 2018; 9 (e02321-18)Crossref Scopus (51) Google Scholar, Pawluk et al., 2016Pawluk A. Amrani N. Zhang Y. Garcia B. Hidalgo-Reyes Y. Lee J. Edraki A. Shah M. Sontheimer E.J. Maxwell K.L. et al.Naturally Occurring Off-Switches for CRISPR-Cas9.Cell. 2016; 167: 1829-1838.e1829Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, Rauch et al., 2017Rauch B.J. Silvis M.R. Hultquist J.F. Waters C.S. McGregor M.J. Krogan N.J. Bondy-Denomy J. Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.Cell. 2017; 168: 150-158.e110Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, Shin et al., 2017Shin J. Jiang F. Liu J.J. Bray N.L. Rauch B.J. Baik S.H. Nogales E. Bondy-Denomy J. Corn J.E. Doudna J.A. Disabling Cas9 by an anti-CRISPR DNA mimic.Sci. Adv. 2017; 3: e1701620Crossref PubMed Scopus (205) Google Scholar). They have also been used to control gene activation (CRISPRa) and gene interference (CRISPRi) in yeast and mammalian cells (Nakamura et al., 2019Nakamura M. Srinivasan P. Chavez M. Carter M.A. Dominguez A.A. La Russa M. Lau M.B. Abbott T.R. Xu X. Zhao D. et al.Anti-CRISPR-mediated control of gene editing and synthetic circuits in eukaryotic cells.Nat. Commun. 2019; 10: 194Crossref PubMed Scopus (84) Google Scholar) and to decrease the toxicity of CRISPR-Cas9 delivered by an adenovirus vector to human stem cells (Li et al., 2018Li C. Psatha N. Gil S. Wang H. Papayannopoulou T. Lieber A. HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic Stem Cells.Mol. Ther. Methods Clin. Dev. 2018; 9: 390-401Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Since the methods of in vivo delivery for CRISPR-Cas9, which include viral vectors and nanoparticles, do not have high tissue specificity, it is crucial to avoid editing in non-targeted tissues, which would increase the risk of unwanted side effects (Cox et al., 2015Cox D.B. Platt R.J. Zhang F. Therapeutic genome editing: prospects and challenges.Nat. Med. 2015; 21: 121-131Crossref PubMed Scopus (838) Google Scholar). Recently, a Cas9-ON switch based on microRNA-dependent expression of an anti-CRISPR protein was used to control gene editing in a cell-specific manner (Hoffmann et al., 2019Hoffmann M.D. Aschenbrenner S. Grosse S. Rapti K. Domenger C. Fakhiri J. Mastel M. Börner K. Eils R. Grimm D. Niopek D. Cell-specific CRISPR-Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins.Nucleic Acids Res. 2019; 47: e75Crossref PubMed Scopus (54) Google Scholar), including in the tissues of adult mice in vivo (Lee et al., 2019Lee J. Mou H. Ibraheim R. Liang S.Q. Liu P. Xue W. Sontheimer E.J. Tissue-restricted Genome Editing in vivo Specified by MicroRNA-repressible Anti-CRISPR Proteins.RNA. 2019; 25: 1421-1431Crossref PubMed Scopus (38) Google Scholar). These applications of anti-CRISPRs are varied, and their potential for further development is enormous. While many different Cas9 proteins exist in nature, only a few are commonly used for genome engineering applications. These include the type II-A Cas9 proteins derived from Streptococcus pyogenes (SpyCas9) and Staphylococcus aureus (SauCas9) (Colella et al., 2017Colella P. Ronzitti G. Mingozzi F. Emerging Issues in AAV-Mediated In Vivo Gene Therapy.Mol. Ther. Methods Clin. Dev. 2017; 8: 87-104Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, Ran et al., 2015Ran F.A. Cong L. Yan W.X. Scott D.A. Gootenberg J.S. Kriz A.J. Zetsche B. Shalem O. Wu X. Makarova K.S. et al.In vivo genome editing using Staphylococcus aureus Cas9.Nature. 2015; 520: 186-191Crossref PubMed Scopus (1780) Google Scholar) and the type II-C Cas9 proteins from Neisseria meningitidis (Nme1Cas9) and Campylobacter jejuni (CjeCas9) (Ibraheim et al., 2018Ibraheim R. Song C.Q. Mir A. Amrani N. Xue W. Sontheimer E.J. All-in-one adeno-associated virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo.Genome Biol. 2018; 19: 137Crossref PubMed Scopus (57) Google Scholar, Kim et al., 2017Kim E. Koo T. Park S.W. Kim D. Kim K. Cho H.Y. Song D.W. Lee K.J. Jung M.H. Kim S. et al.In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni.Nat. Commun. 2017; 8: 14500Crossref PubMed Scopus (406) Google Scholar, Lee et al., 2016Lee C.M. Cradick T.J. Bao G. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells.Mol. Ther. 2016; 24: 645-654Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, Mir et al., 2018bMir A. Edraki A. Lee J. Sontheimer E.J. Type II-C CRISPR-Cas9 Biology, Mechanism, and Application.ACS Chem. Biol. 2018; 13: 357-365Crossref PubMed Scopus (52) Google Scholar, Zhang et al., 2015Zhang Y. Rajan R. Seifert H.S. Mondragón A. Sontheimer E.J. DNase H Activity of Neisseria meningitidis Cas9.Mol. Cell. 2015; 60: 242-255Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). These Cas9 homologs vary in features such as protospacer adjacent motif (PAM) specificity, size, and off-target activity, which makes each more or less advantageous for particular genome-editing applications. Anti-CRISPRs that target some of these Cas9 proteins have been identified (Harrington et al., 2017Harrington L.B. Doxzen K.W. Ma E. Liu J.J. Knott G.J. Edraki A. Garcia B. Amrani N. Chen J.S. Cofsky J.C. et al.A Broad-Spectrum Inhibitor of CRISPR-Cas9.Cell. 2017; 170: 1224-1233.e1215Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, Hynes et al., 2017Hynes A.P. Rousseau G.M. Lemay M.L. Horvath P. Romero D.A. Fremaux C. Moineau S. An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9.Nat. Microbiol. 2017; 2: 1374-1380Crossref PubMed Scopus (111) Google Scholar, Pawluk et al., 2016Pawluk A. Amrani N. Zhang Y. Garcia B. Hidalgo-Reyes Y. Lee J. Edraki A. Shah M. Sontheimer E.J. Maxwell K.L. et al.Naturally Occurring Off-Switches for CRISPR-Cas9.Cell. 2016; 167: 1829-1838.e1829Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, Rauch et al., 2017Rauch B.J. Silvis M.R. Hultquist J.F. Waters C.S. McGregor M.J. Krogan N.J. Bondy-Denomy J. Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.Cell. 2017; 168: 150-158.e110Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar), but none of these efficiently inhibit all of them. The identification of a well-characterized, universal anti-CRISPR protein that could function to control Cas9 activity in a variety of different applications—including genome editing, gene drives, and CRISPRi/CRISPRa—would have broad utility and could hasten the development of these technologies. Thus, the goal of this work was to identify an anti-CRISPR with broad and potent activity. In this study, we investigated the spectra of inhibition of a variety of previously described anti-CRISPRs that showed activity against type II-A (Hynes et al., 2018Hynes A.P. Rousseau G.M. Agudelo D. Goulet A. Amigues B. Loehr J. Romero D.A. Fremaux C. Horvath P. Doyon Y. et al.Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins.Nat. Commun. 2018; 9: 2919Crossref PubMed Scopus (98) Google Scholar, Hynes et al., 2017Hynes A.P. Rousseau G.M. Lemay M.L. Horvath P. Romero D.A. Fremaux C. Moineau S. An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9.Nat. Microbiol. 2017; 2: 1374-1380Crossref PubMed Scopus (111) Google Scholar, Rauch et al., 2017Rauch B.J. Silvis M.R. Hultquist J.F. Waters C.S. McGregor M.J. Krogan N.J. Bondy-Denomy J. Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.Cell. 2017; 168: 150-158.e110Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, Uribe et al., 2019Uribe R.V. van der Helm E. Misiakou M.A. Lee S.W. Kol S. Sommer M.O.A. Discovery and Characterization of Cas9 Inhibitors Disseminated across Seven Bacterial Phyla.Cell Host Microbe. 2019; 25: 233-241.e235Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and type II-C (Mir et al., 2018bMir A. Edraki A. Lee J. Sontheimer E.J. Type II-C CRISPR-Cas9 Biology, Mechanism, and Application.ACS Chem. Biol. 2018; 13: 357-365Crossref PubMed Scopus (52) Google Scholar, Pawluk et al., 2016Pawluk A. Amrani N. Zhang Y. Garcia B. Hidalgo-Reyes Y. Lee J. Edraki A. Shah M. Sontheimer E.J. Maxwell K.L. et al.Naturally Occurring Off-Switches for CRISPR-Cas9.Cell. 2016; 167: 1829-1838.e1829Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) CRISPR-Cas9 systems using an efficient E. coli phage-based assay system. We discovered that the previously identified anti-CRISPR, AcrIIA5 from Streptococcus thermophilus phage D4276 (Hynes et al., 2017Hynes A.P. Rousseau G.M. Lemay M.L. Horvath P. Romero D.A. Fremaux C. Moineau S. An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9.Nat. Microbiol. 2017; 2: 1374-1380Crossref PubMed Scopus (111) Google Scholar), has the broadest Cas9 inhibitory activity described to date, inhibiting all of the Cas9 proteins commonly used in genome-editing applications. A key initial goal of this work was to develop a system to identify anti-CRISPRs with the broadest possible spectrum of activity for use in Cas9-based technologies. To quantitatively compare the specificity profiles of a large number of anti-CRISPR proteins, we expanded upon a previously described phage-targeting assay in which Cas9 from Geobacillus stearothermophilus (GeoCas9) was engineered to prevent infection by E. coli phage Mu (Harrington et al., 2017Harrington L.B. Doxzen K.W. Ma E. Liu J.J. Knott G.J. Edraki A. Garcia B. Amrani N. Chen J.S. Cofsky J.C. et al.A Broad-Spectrum Inhibitor of CRISPR-Cas9.Cell. 2017; 170: 1224-1233.e1215Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). In this assay, GeoCas9 was co-expressed with an sgRNA that targets phage Mu and prevents its replication by cleaving its genome. In the current work, we expressed a diverse group of Cas9 homologs (Figures 1A and 1B ) in E. coli, each engineered to target phage Mu. These Cas9 homologs include those commonly used in genome-editing applications, including SpyCas9, SauCas9, CjeCas9, and Nme1Cas9. We also chose six additional Cas9 homologs distributed across the phylogeny of Cas9s occurring in bacteria (Figure 1A). All three subtypes (II-A, II-B, and II-C) were represented among these Cas9s, which range in pairwise sequence identity from 19% to 66% and utilize a variety of PAM sequences (Figure 1B). We tested 10 previously identified anti-CRISPRs in the phage Mu targeting assay, including four that were shown to inhibit type II-A and five that inhibit type II-C CRISPR-Cas systems. As seen in Figure 1C, the targeted cleavage activity of each of these Cas9 proteins reduced the plaquing efficiency of phage Mu by at least 105-fold compared to strains expressing the same Cas9 proteins with non-targeting sgRNA (Figure 1C). The co-expression of anti-CRISPRs completely reversed the Cas9-mediated reduction of plaquing efficiency in some cases (Figure 1C). However, in other cases, anti-CRISPR co-expression caused no increase or only a partial increase in plaquing efficiency. The level of phage Mu plaquing in the presence of a particular Cas9/anti-CRISPR combination provides a quantitative measure of the effectiveness of the anti-CRISPR in inhibiting a given Cas9 homolog. Some anti-CRISPRs, such as AcrIIA4, are very specific, inhibiting only one or a few CRISPR-Cas9 systems, while others, such as AcrIIC1, strongly inhibited many different Cas9s (Figure 1D). Overall, the results in Figure 1D show that the strength of anti-CRISPRs may vary over many orders of magnitude, and the specificity profile of each anti-CRISPR is unique. In contrast to all of the other anti-CRISPRs tested, AcrIIA5 was able to completely inhibit every type II-A and II-C Cas9 tested, failing to block only the type II-B Cas9 from Francisella novicida (Figures 1C and 1D). AcrIIA5 was the only anti-CRISPR able to block the highly divergent CdiCas9, emphasizing its unusually broad activity. A previous in vitro study noted the ability of AcrIIA5 to inhibit CjeCas9 and a homolog of AcrIIA5 to inhibit Nme1Cas9 (Marshall et al., 2018Marshall R. Maxwell C.S. Collins S.P. Jacobsen T. Luo M.L. Begemann M.B. Gray B.N. January E. Singer A. He Y. et al.Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System.Mol. Cell. 2018; 69: 146-157.e143Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The uniquely broad specificity of AcrIIA5 inspired us to further investigate its properties. Although AcrIIA5 was previously shown to inhibit genome editing mediated by SpyCas9 and Streptococcus thermophilus Cas9 (St1Cas9) in mammalian cells (Hynes et al., 2017Hynes A.P. Rousseau G.M. Lemay M.L. Horvath P. Romero D.A. Fremaux C. Moineau S. An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9.Nat. Microbiol. 2017; 2: 1374-1380Crossref PubMed Scopus (111) Google Scholar), its activity against other Cas9 proteins in genome-editing applications had not been tested. To determine if AcrIIA5 could inhibit genome editing mediated by the four Cas9 homologs commonly used for genome-editing purposes in mammalian cells, we transiently co-transfected mouse Neuro-2a (N2a) (Figure 2A) or human HEK293T (Figure 2B) cells with plasmids expressing anti-CRISPR proteins, Cas9s and their respective sgRNAs designed to target specific genomic sites. Tracking of indels by decomposition (TIDE) analyses (Brinkman et al., 2014Brinkman E.K. Chen T. Amendola M. van Steensel B. Easy quantitative assessment of genome editing by sequence trace decomposition.Nucleic Acids Res. 2014; 42: e168Crossref PubMed Scopus (1163) Google Scholar) revealed that AcrIIA5 inhibited the activities of SpyCas9, Nme1Cas9, SauCas9, and CjeCas9. These results were confirmed using a previously described T7 endonuclease I (T7E1) assay (Figure 2C) (Pawluk et al., 2016Pawluk A. Amrani N. Zhang Y. Garcia B. Hidalgo-Reyes Y. Lee J. Edraki A. Shah M. Sontheimer E.J. Maxwell K.L. et al.Naturally Occurring Off-Switches for CRISPR-Cas9.Cell. 2016; 167: 1829-1838.e1829Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). We further probed the ability of AcrIIA5 to inhibit Cas9 homologs using a variation of the traffic light reporter (TLR) system (Certo et al., 2011Certo M.T. Ryu B.Y. Annis J.E. Garibov M. Jarjour J. Rawlings D.J. Scharenberg A.M. Tracking genome engineering outcome at individual DNA breakpoints.Nat. Methods. 2011; 8: 671-676Crossref PubMed Scopus (214) Google Scholar), which contains an artificial locus harboring Cas9 target sites. In this assay, an out-of-frame mCherry gene is targeted for Cas9 editing, resulting in a subset of indels that restore the proper reading frame for mCherry, thereby generating a fluorescent signal. Co-transfection of Cas9 homologs and their respective sgRNAs targeting the TLR locus resulted in cells with mCherry expression ranging from 5% to 20%, depending on the Cas9 used for editing. Expression of AcrIIA5 by transient transfection reduced the editing at the TLR locus by all of the Cas9 homologs tested (Figure 2D). Collectively, these results show that AcrIIA5 efficiently inhibits the in vivo genome-editing activity of four diverse Cas9 proteins in both bacterial and mammalian cells. Furthermore, AcrIIA5 inhibits genome editing with similar potency to previously utilized anti-CRISPRs. To investigate how AcrIIA5 inhibits Cas9 activity, we developed a luminescence-based bioassay in which we targeted the catalytically inactive dSpyCas9 (Gilbert et al., 2014Gilbert L.A. Horlbeck M.A. Adamson B. Villalta J.E. Chen Y. Whitehead E.H. Guimaraes C. Panning B. Ploegh H.L. Bassik M.C. et al.Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation.Cell. 2014; 159: 647-661Abstract Full Text Full Text PDF PubMed Scopus (1557) Google Scholar) to a constitutively expressed artificial promoter that drives expression of the luxCDABE luminescence genes in E. coli (Figure 3A). sgRNA-targeted binding of dSpyCas9 to the promoter of the luxCDABE operon repressed transcription, and no luminescence was detected (Figure 3B). Expression of AcrIIA5 relieved this repression, leading to an increase in luminescence and showing that DNA binding was inhibited. Similarly, expression of AcrIIA4, which was previously shown to inhibit SpyCas9 DNA binding (Dong et al., 2017Dong D. Guo M. Wang S. Zhu Y. Wang S. Xiong Z. Yang J. Xu Z. Huang Z. Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRISPR protein.Nature. 2017; 546: 436-439Crossref PubMed Scopus (151) Google Scholar, Shin et al., 2017Shin J. Jiang F. Liu J.J. Bray N.L. Rauch B.J. Baik S.H. Nogales E. Bondy-Denomy J. Corn J.E. Doudna J.A. Disabling Cas9 by an anti-CRISPR DNA mimic.Sci. Adv. 2017; 3: e1701620Crossref PubMed Scopus (205) Google Scholar, Yang and Patel, 2017Yang H. Patel D.J. Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9.Mol. Cell. 2017; 67: 117-127.e115Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), also led to an increase in luminescence. By contrast, expression of AcrIIC1, which does not inhibit SpyCas9 (Harrington et al., 2017Harrington L.B. Doxzen K.W. Ma E. Liu J.J. Knott G.J. Edraki A. Garcia B. Amrani N. Chen J.S. Cofsky J.C. et al.A Broad-Spectrum Inhibitor of CRISPR-Cas9.Cell. 2017; 170: 1224-1233.e1215Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, Pawluk et al., 2016Pawluk A. Amrani N. Zhang Y. Garcia B. Hidalgo-Reyes Y. Lee J. Edraki A. Shah M. Sontheimer E.J. Maxwell K.L. et al.Naturally Occurring Off-Switches for CRISPR-Cas9.Cell. 2016; 167: 1829-1838.e1829Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar), showed no increase in luminescence, as expected. These results demonstrate that AcrIIA5 blocks binding of dSpyCas9 to target DNA and impedes its function as a transcriptional repressor. After co-expression of His6-tagged Nme1Cas9 and AcrIIA5, AcrIIA5 did not co-elute with Nme1Cas9, while a control, AcrIIC1, did co-elute (Figure 3C). Nevertheless, Nme1Cas9 expressed in the presence of AcrIIA5 was unable to cleave DNA in vitro (Figure 3D). Thus, co-expression of AcrIIA5 with Nme1Cas9 caused a loss of activity even though the anti-CRISPR did not form a stable complex with Cas9. Electrophoretic examination of the sgRNA bound to Nme1Cas9 purified in the presence of AcrIIA5 surprisingly showed that a sizable proportion was smaller compared to the sgRNA bound to Nme1Cas9 expressed without AcrIIA5 or with AcrIIC1 (Figures 3C, S1A, and S1B). The full-length and cleaved sgRNA molecules seen in these gels were excised, reverse transcribed into DNA, and sequenced. We found that a portion of the Nme1Cas9 co-expressed with AcrIIA5 was bound to full-length sgRNA that was indistinguishable from that of Nme1Cas9 controls, but it was also frequently bound to truncated forms (Figure S1B). These truncations mapped to stem-loop 1 and stem-loop 2 of the sgRNA (Figures 3E and S1C). It was recently shown that Nme1Cas9 can mediate RNA cleavage that is catalyzed by the Cas9 HNH endonuclease domain (Rousseau et al., 2018Rousseau B.A. Hou Z. Gramelspacher M.J. Zhang Y. Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis.Mol. Cell. 2018; 69: 906-914.e904Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). However, the formation of the truncated sgRNA molecules seen here was not mediated by either of the nuclease domains of Nme1Cas9 (Figure S1D). To investigate the relationship between AcrIIA5 activity and CRISPR RNA (crRNA) in a genome-editing application in mammalian cells, we tested its ability to inhibit editing efficiency in the presence of chemically modified crRNA/trans-activating crRNA (tracrRNA) molecules that were previously described (Mir et al., 2018aMir A. Alterman J.F. Hassler M.R. Debacker A.J. Hudgens E. Echeverria D. Brodsky M.H. Khvorova A. Watts J.K. Sontheimer E.J. Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing.Nat. Commun. 2018; 9: 2641Crossref PubMed Scopus (51) Google Scholar). In a cell line that stably expresses AcrIIA5, editing efficiency was completely abrogated when an unmodified guide (C0:T0) was used (see STAR Methods for details of the modified RNA molecules) but was only partially compromised (∼3-fold) when an RNase-resistant heavily modified guide (C20:T2) was used (Figure 3F). The protection from AcrIIA5 activity provided by chemical modification of the crRNA/tracrRNA is consistent with this anti-CRISPR interacting with RNA. A caveat to this experiment is that the unmodified RNA mediated genome editing much less efficiently (∼5-fold) than the modified RNA. However, given that TIDE methodology can detect editing levels of ∼1%–2% (Brinkman et al., 2014Brinkman E.K. Chen T. Amendola M. van Steensel B. Easy quantitative assessment of genome editing by sequence trace decomposition.Nucleic Acids Res. 2014; 42: e168Crossref PubMed Scopus (1163) Google Scholar), and editing by Cas9 bound to unmodified RNA was undetectable, we estimate that AcrIIA5 reduced editing mediated by unmodified RNA by at least 15-fold, while that mediated by modified RNA was only reduced by ∼3-fold. Despite extensive efforts, AcrIIA5 could not be purified in a soluble and active form on its own. In an effort to circumvent this problem, we also cloned and expressed five additional AcrIIA5 family members (Figures S2A and S2B), which ranged from 87% to 48% in sequence identity at the amino acid level from the homolog from S. thermophilus phage D4276 that we characterized here. Although each of these homologs robustly inhibited all Cas9s tested (Figure S2C) and were also well expressed in E. coli (Figure S2D), none could be purified in a soluble and active form. Thus, we were unable to carry out the detailed in vitro experiments necessary to further elucidate the AcrIIA5 inhibitory mechanism. To address the question of whether sgRNA cleavage is a consistent feature of the AcrIIA5 family, we purified Nme1Cas9 co-expressed with each of the five AcrIIA5 homologs described above. Notably, the sgRNA co-purifying in these Nme1Cas9 preparations displayed similar levels of partial cleavage in every case (Figure S2E). To establish that sgRNA cleavage was not due to a unique feature of Nme1Cas9, we also co-expressed the AcrIIA5 homologs with SpyCas9 and purified the resulting Cas9/sgRNA complexes. The sgRNA molecules associated with SpyCas9 were also cleaved in the presence of each of the AcrIIA5 homologs (Figure S2F). Finally, we constructed three AcrIIA5 mutants bearing amino acid substitutions at residues potentially involved in catalysis (Figure S2A). Two of these mutants displayed no inhibitory activity against all tested Cas9 homologs (Figures S3A and S3B). Interestingly, the fourth mutant (H66N70H73) was fully active against SpyCas9 but was unable to inhibit the other Cas9 homologs. This indicates that there may be distinct regions of AcrIIA5 responsible for binding to different Cas9 homologs. Purified Nme1Cas9 that was co-expressed with the inactive mutants was not associated with cleaved sgRNA, supporting the connection between AcrIIA5 inhibition of Cas9 and sgRNA cleavage (Figure S3C). AcrIIA5 is a remarkably broad specificity anti-CRISPR that functions through a unique mechanism. The co-expression of AcrIIA5 with Nme1Cas9 results in the truncation of the sgRNA from the 3′ end. This sgRNA truncation was seen consistently in six different AcrIIA5 family members when co-expressed with Nme1Cas9 or SpyCas9. The enigmatic feature of this sgRNA truncation is that the relative amounts of truncated products vary in different experiments, as do the apparent numbers of truncated products (i.e., the number of higher-mobility bands seen in the gels) (Figures 3C, S1B, S1D, S2E, S2F, and S3C). In addition, there is always some sgRNA remaining bound to Cas9 co-expressed with AcrIIA5 that displays the mobility of full-length sgRNA in denaturing polyacrylamide/Urea gels, and sequencing confirmed that this sgRNA is indistinguishable from that bound to Nme1Cas9 in the absence of AcrIIA5 (Figure S1C). We conclude that sgRNA cleavage alone cannot account for the potent inhibitory activity of AcrIIA5. Rather, the action of AcrIIA5 may partially dislodge the sgRNA from Cas9, leaving it prone to digestion by intracellular RNases. The portion of the sgRNA that we observe to be digested, stem-loops 1 and 2, are the more exposed parts of the sgRN" @default.
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• W2987201345 date "2019-11-01" @default.
• W2987201345 modified "2023-10-10" @default.
• W2987201345 title "Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing" @default.
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• W2987201345 doi "https://doi.org/10.1016/j.celrep.2019.10.017" @default.
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