Solution structure and dynamics of anti-CRISPR AcrIIA4, the Cas9 inhibitor

Title
Solution structure and dynamics of anti-CRISPR AcrIIA4, the Cas9 inhibitor
Authors
김낙균Iktae KimMigyeong JeongMookyoung HanEuiyoung BaeJeong-Yong SuhDonghyun Ka
Keywords
CRISPR; Cas9 inhibitor; AcrIIA4; structure; dynamics
Issue Date
2018-03
Publisher
Scientific Reports
Citation
VOL 8, NO 1-3883-9
Abstract
The bacterial CRISPR-Cas system provides adaptive immunity against invading phages. Cas9, an RNA-guided endonuclease, specifically cleaves target DNA substrates and constitutes a well-established platform for genome editing. Recently, anti-CRISPR (Acr) proteins that inhibit Cas9 have been discovered, promising a useful off-switch for Cas9 to avoid undesirable off-target effects. Here, we report the solution structure and dynamics of Listeria monocytogenes AcrIIA4 that inhibits Streptococcus pyogenes Cas9 (SpyCas9). AcrIIA4 forms a compact monomeric alpha beta beta beta alpha alpha fold comprising three antiparallel beta strands flanked by three alpha-helices and a short 3(10)-helix. AcrIIA4 exhibits distinct backbone dynamics in fast and slow timescales at loop regions that form interaction surfaces for SpyCas9. In particular, the beta 1-beta 2 loop that binds to the RuvC domain of SpyCas9 is highly mobile, and the beta 1-beta 2 and alpha 2-alpha 3 loops that bind to the RuvC and C-terminal domains of SpyCas9, respectively, undergoes conformational exchanges in microsecond-to-millisecond time scales. AcrIIA4 binds to apo-SpyCas9 with K-D similar to 4.8 mu M, which compares to K-D similar to 0.6 nM for AcrIIA4 binding to sgRNA-bound SpyCas9. Since the binary complex between AcrIIA4 and SpyCas9 does not compete with the target DNA binding, it can effectively disable the Cas9 nuclease activity by forming a tight ternary complex in the presence of sgRNA.
URI
http://pubs.kist.re.kr/handle/201004/68471
ISSN
2045-2322
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