Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins

Hynes, Alexander P.; Rousseau, Geneviève M.; Agudelo, Daniel; Goulet, Adeline; Amigues, Béatrice; Loehr, Jérémy; Romero, Dennis; Fremaux, Christophe; Horvath, Philippe; Doyon, Yannick; Cambillau, Christian; Moineau, Sylvain

doi:10.1038/s41467-018-05092-w

Cited by 157 publications

(162 citation statements)

References 62 publications

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“…We next tested three known Acrs for their ability to inhibit SauCas9-mediated genome editing in mammalian cells, including the robust SpyCas9 inhibitor AcrIIA4 (10), the broadspectrum type II-A Cas9 inhibitor AcrIIA5 (12) and the species-specific Streptococcus thermophilus Cas9 inhibitor AcrIIA6 (12). While AcrIIA4 prevented genome editing by SpyCas9 as expected (10), we found that it had no effect against SauCas9 (Fig.…”

Section: Inhibition Of Saucas9-mediated Genome Editing In Human Cellsmentioning

confidence: 59%

“…However, some MGEs, particularly phages, have evolved anti-CRISPRs (Acrs), peptide inhibitors of Cas proteins that block CRISPR defense systems (2,3). Acrs have been discovered to inhibit distinct CRISPR systems, including type I (4)(5)(6)(7)(8), type II (9)(10)(11)(12)(13)(14)(15)(16), type III (17,18), and type V (7,19). Strategies for identifying new Acrs include testing genes of unknown function that are proximal to anti-CRISPR associated (aca) genes (5-7, 9, 15) and screening genes in organisms with self-targeting CRISPR systems (7,19,20).…”

Section: Introductionmentioning

confidence: 99%

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Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Watters

Shivram

Fellmann

et al. 2019

Preprint

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2Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Casmediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three new potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14 and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for anti-CRISPR function, and have divergent C-termini that are required in each case for selective inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust and specific inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-15 as unique bifunctional inhibitors of SauCas9.

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Section: Inhibition Of Saucas9-mediated Genome Editing In Human Cellsmentioning

confidence: 59%

Section: Introductionmentioning

confidence: 99%

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Watters

Shivram

Fellmann

et al. 2019

Preprint

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“…While characterizing the interplay between St1Cas9 and diverse Acr families isolated from virulent phages infecting S. thermophilus 31 , we were surprised by the substantial levels of editing achieved in human cells 33 . This observation contrasts with early reports indicating that this ortholog was mildly active 7,34 .…”

Section: Identification Of An Sgrna Architecture Directing Robust Dnamentioning

confidence: 98%

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Agudelo

Carter

Velimirovic

et al. 2018

Preprint

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The broad spectrum of activities displayed by CRISPR-Cas systems has led to biotechnological innovations that are poised to transform human therapeutics. Therefore, the comprehensive characterization of distinct Cas proteins is highly desirable. Here we expand the repertoire of nucleases for mammalian genome editing using the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9). We define functional protospacer adjacent motif (PAM) sequences and variables required for robust and efficient editing in vitro. Expression of holoSt1Cas9 from a single adeno-associated viral (rAAV) vector in the neonatal liver rescued lethality and metabolic defects in a mouse model of hereditary tyrosinemia type I demonstrating effective cleavage activity in vivo. Furthermore, we identified potent anti-CRISPR proteins to regulate the activity of both St1Cas9 and the related type II-A Staphylococcus aureus Cas9 (SaCas9). This work expands the targeting range and versatility of CRISPR-associated enzymes and should encourage studies to determine its structure, genome-wide specificity profile and sgRNA design rules. INTRODUCTIONClustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins form a prokaryotic adaptive immune system and some of its components have been harnessed for robust genome editing 1 . Type II-based editing tools rely on a large multidomain endonuclease, Cas9, guided to its DNA target by an engineered single-guide RNA (sgRNA) chimera 2 (See 3, 4 for a classification of CRISPR-Cas systems). The Cas9-sgRNA binary complex finds its target through recognition of a short sequence called the protospacer adjacent motif (PAM) and subsequent base pairing of the guide RNA with the DNA to generate a specific doublestrand break (DSB) 1,5 . While Streptococcus pyogenes (SpCas9) remains the most widely used Cas9 variant for genome engineering, the diversity of naturally occurring RNA-guided nucleases is astonishing 4 . Hence, Cas9 enzymes from different microbial species can contribute to the expansion of the CRISPR toolset by increasing targeting density, improving activity and specificity as well as easing delivery 1,6 .In principle, engineering complementary CRISPRCas systems from distinct bacterial species should be relatively straightforward, as they have been minimized to only two components. However, many such enzymes were found inactive in human cells despite being accurately reprogrammed for DNA binding and cleavage in vitro [7][8][9][10] . Nevertheless, the full potential of selected enzymes can be unleashed using machine learning to establish sgRNA design rules 11,12 .Perhaps the most striking example of the value of alternative Cas9 enzymes is the implementation of the type II-A Cas9 from Staphylococcus aureus (SaCas9) for in vivo editing using recombinant adeno-associated virus (rAAV) vectors 7,13, 14 . More recently, Campylobacter jejuni and Neisseria meningitidis Cas9s from the type II-C 15 CRISPRCas systems have been added to this repertoire 16,17 .In vivo genome edi...

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“…A number of Acrs have been identified for type I [23,[25][26][27][28], type II [29][30][31][32] and type V [28,33] CRISPR-Cas systems. Acrs can be adapted to regulate CRISPR-Cas activities in bacteria [34], yeast [35] and mammalian cells [29,31,34,[36][37][38]. Biosensor [39] and synthetic circuits [40] can be devised based on Acr-coupled CRISPR-Cas systems.…”

Section: Inhibition Off-target Activitymentioning

confidence: 99%

Allosteric Inhibition of CRISPR-Cas9 by Bacteriophage-derived Peptides

Cui

Wang

et al. 2019

Preprint

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Background CRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system.Excessive and prolonged expression CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over the dimensions of time and dose has created the demand of developing CRISPR-Cas off-switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies. ResultsWe report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpyCas9) proteins in an allosteric manner. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpyCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpyCas9 while retaining its on-target activity. ConclusionG8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.

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Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins

Cited by 157 publications

References 62 publications

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Allosteric Inhibition of CRISPR-Cas9 by Bacteriophage-derived Peptides

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