Anti‐CRISPRs: The natural inhibitors for CRISPR‐Cas systems

Zhang, Fei; Song, Guoxu; Tian, Yong

doi:10.1002/ame2.12069

Cited by 32 publications

(25 citation statements)

References 73 publications

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“…Numerous strategies continue to be developed for identification of Acrs, with a remarkably diverse range of disclosed inhibition mechanisms 33 , 34 . Here, we discovered unusual acr loci in various MGEs, led initially by gene associations with acrIIA1 19 , 31 .…”

Section: Discussionmentioning

confidence: 99%

Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer

et al. 2020

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CRISPR-Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes , using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus, and Staphylococcus genomes, can inhibit Type II-A SpyCas9 or SauCas9, and are thus named acrIIA16 - 19 . In Enterococcus faecalis, conjugation of a Cas9-targeted plasmid was enhanced by anti-CRISPRs derived from Enterococcus conjugative elements, highlighting a role for Acrs in the dissemination of plasmids. Reciprocal co-immunoprecipitation showed that each Acr protein interacts with Cas9, and Cas9:Acr complexes were unable to cleave DNA. Northern blotting suggests that these anti-CRISPRs manipulate sgRNA loading or stability. Mirroring their activity in bacteria, AcrIIA16 and AcrIIA17 provide robust and highly potent broad-spectrum inhibition of distinct Cas9 proteins in human cells (e.g. SpyCas9, SauCas9, SthCas9, NmeCas9, CjeCas9). This work presents a focused analysis of non-phage Acr proteins, demonstrating a role in horizontal gene transfer bolstered by broad spectrum CRISPR-Cas9 inhibition.

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Section: Discussionmentioning

confidence: 99%

Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer

et al. 2020

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“…These CRISPR antagonists are diverse and widespread among phages for the large diversity of CRISPR/Cas systems [ 29 , 30 ]. Currently, forty-four unique families of anti-CRISPR proteins have been reported in roles against both class 1 and class 2 CRISPR/Cas systems [ 31 ]. Remarkably, of these inhibitors, AcrIIA2, AcrIIA4, and AcrIIA5 could be used to inhibit type II-A Streptococcus pyogenes Cas9 (SpyCas9)-mediated gene editing [ 28 , 32 , 33 ].…”

Section: Introductionmentioning

confidence: 99%

AcrIIA5 Suppresses Base Editors and Reduces Their Off-Target Effects

Liang

Sui

Liu

et al. 2020

Cells

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The CRISPR/nCas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) are capable of catalyzing C•G to T•A or A•T to G•C conversions, respectively, and have become new, powerful tools for achieving precise genetic changes in a wide range of organisms. These base editors hold great promise for correcting pathogenic mutations and for being used for therapeutic applications. However, the recognition of cognate DNA sequences near their target sites can cause severe off-target effects that greatly limit their clinical applications, and this is an urgent problem that needs to be resolved for base editing systems. The recently discovered phage-derived proteins, anti-CRISPRs, which can suppress the natural CRISPR nuclease activity, may be able to ameliorate the off-target effects of base editing systems. Here, we confirm for the first time that AcrIIA2, AcrIIA4, and AcrIIA5 efficiently inhibit base editing systems in human cells. In particular, AcrIIA5 has a significant inhibitory effect on all base editing variant systems tested in our study. We further show that the off-target effects of BE3 and ABE7.10 were significantly reduced in AcrIIA5 treated cells. This study suggests that AcrIIA5 should be widely used for the precise control of base editing and to thoroughly “shut off” nuclease activity of both CBE and ABE systems.

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“…CRISPR systems are RNA-guided, adaptive immune systems that defend prokaryotes against invading mobile genetic elements (MGEs) (1). 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).…”

Section: Introductionmentioning

confidence: 99%

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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Anti‐CRISPRs: The natural inhibitors for CRISPR‐Cas systems

Cited by 32 publications

References 73 publications

Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer

Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer

AcrIIA5 Suppresses Base Editors and Reduces Their Off-Target Effects

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

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