2017
DOI: 10.1016/j.cell.2017.07.037
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A Broad-Spectrum Inhibitor of CRISPR-Cas9

Abstract: SUMMARY CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off-switch for Cas9-based applications. Here we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through dir… Show more

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Cited by 234 publications
(375 citation statements)
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References 65 publications
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“…The steric clash with AcrIIC3 was more severe in the complex structure with the PAM duplex than that with the full dsDNA, implying that AcrIIC3 might interfere with conformational changes of Cas9 required for sgRNA–DNA hybridization after PAM recognition. The steric hindrance of AcrIIC3 is consistent with the previous observation that AcrIIC3 reduced the DNA binding of NmeCas9 by ~ 10‐fold .…”
Section: Discussionsupporting
confidence: 90%
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“…The steric clash with AcrIIC3 was more severe in the complex structure with the PAM duplex than that with the full dsDNA, implying that AcrIIC3 might interfere with conformational changes of Cas9 required for sgRNA–DNA hybridization after PAM recognition. The steric hindrance of AcrIIC3 is consistent with the previous observation that AcrIIC3 reduced the DNA binding of NmeCas9 by ~ 10‐fold .…”
Section: Discussionsupporting
confidence: 90%
“…2B). This affinity was comparable to the K D of~6.3 AE 3.4 nM measured between AcrIIC1 and NmeHNH [19]. We determined the crystal structure of AcrIIC3 bound to NmeHNH at a resolution of 2.0 A ( Table 1).…”
Section: Acriic3 Targets the Cas9 Hnh Domain In An Allosteric Mannersupporting
confidence: 60%
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“…Interestingly, the ways in which AcrIIC1 and AcrIIC3 inhibit Cas9 target binding and cleavage are quite different. AcrIIC1 is an extensively used Cas9 inhibitor that inhibits many distinct Cas9 proteins by binding directly to the HNH nuclease domain, which is highly conserved among all Cas9 proteins, without preventing DNA binding . Conversely, AcrIIC3 inhibits the activity of a single Cas9 ortholog; instead of interacting directly with the HNH nuclease domain of Cas9, it binds two Cas9 proteins to induce Cas9 dimerization, which suggests that the conformation of Cas9 undergoes conformational change or oligomerization such that it loses the ability to bind DNA .…”
Section: Mechanisms and Functions Of Acrsmentioning
confidence: 99%