Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing

Abstract

SUMMARY 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.

Potent CRISPR-Cas9 inhibitors fromStaphylococcusgenomes

“…S7). Conversely, AcrIIA5 displayed strong inhibitory activity toward both SauCas9 and SpyCas9, as recently shown (30), while AcrIIA6 did not show any inhibition of either of the two Cas9s.…”

“…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 broad-spectrum type II-A Cas9 inhibitor AcrIIA5 (12,30), and the speciesspecific 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 (SI Appendix, Fig.…”

“…To determine the stage at which the Acrs are able to inhibit SauCas9 activity, we performed the SauCas9 in vitro cleavage experiments again, but added the Acr proteins before the addition of sgRNA. Adding the Acr protein before the sgRNA led to an approximately fourfold increase in inhibition by the Acr proteins, on par with the only currently known broad spectrum Acr for type II-A Cas9, AcrIIA5 (11,30) (Fig. 3 A, Right and SI Appendix, Fig.…”

Diversity of molecular mechanisms used by anti-CRISPR proteins: the tip of an iceberg?

“…To date, inhibition of target binding is the prevalent strategy. Thirteen anti-CRISPR proteins interfere with target recognition and binding (type I-F AcrIF1, AcrIF2 and AcrIF10 [34][35][36][37]; type II-A AcrIIA2, AcrIIA4, AcrIIA5 and AcrIIA6 [38][39][40][41][42][43][44][45][46][47]; type II-C AcrIIC3, AcrIIC4 and AcrIIC5 [48][49][50][51]; type V-A AcrVA1, AcrV4A and AcrVA5 [52][53][54][55][56][57][58]), while only five-block target cleavage (type I-E AcrIE1 [29,59]; type III-B AcrIIIB1 [12]; type I-F AcrIF3 [26,27]; type II-C AcrIIC1 and AcrIIC3 [48][49][50]) ( Figure 1). Given that DNA binding is the ratelimiting step of Cascade and Cas9-mediated interference activities [60,61], altering this step is, therefore, an efficient way to inactivate CRISPR-Cas interference.…”

“…Noteworthy, AcrVA1 mimics the PAM to position its catalytic residues close to the crRNA substrate [57]. Interestingly, the type II-A AcrIIA5 anti-CRISPR protein was recently shown to lead to sgRNA cleavage at multiples sites out of the crRNA spacer sequence [45]. However, whether or not this anti-CRISPR protein also has a nuclease activity remains to be determined.…”