Journal Article

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

Suh, Jeong-Yong, Han, Mookyoung, Kim, Iktae, Jeong, Migyeong, Kim, Nak-Kyoon, Bae, Euiyoung, Ka, Donghyun

2018

Overview

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 ( Spy Cas9). AcrIIA4 forms a compact monomeric αβββαα fold comprising three antiparallel β strands flanked by three α-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 Spy Cas9. In particular, the β1–β2 loop that binds to the RuvC domain of Spy Cas9 is highly mobile, and the β1–β2 and α2–α3 loops that bind to the RuvC and C-terminal domains of Spy Cas9, respectively, undergoes conformational exchanges in microsecond-to-millisecond time scales. AcrIIA4 binds to apo- Spy Cas9 with K D ~4.8 μM, which compares to K D ~0.6 nM for AcrIIA4 binding to sgRNA-bound Spy Cas9. Since the binary complex between AcrIIA4 and Spy Cas9 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 101/6 / 140/131 / 631/535/878/1263 / 631/57/2272/2273 / Adaptive immunity / Bacterial Proteins - genetics / Bacterial Proteins - metabolism / Bacteriophages - genetics / Clustered Regularly Interspaced Short Palindromic Repeats - physiology / CRISPR / CRISPR-Associated Protein 9 - antagonists & inhibitors / CRISPR-Associated Protein 9 - chemistry / CRISPR-Associated Protein 9 - genetics / CRISPR-Cas Systems / Deoxyribonucleic acid / DNA / DNA - chemistry / Endonuclease / Endonucleases - antagonists & inhibitors / Endonucleases - genetics / Gene Editing - methods / Genome editing / Genomes / Humanities and Social Sciences / Listeria monocytogenes / Listeria monocytogenes - enzymology / Listeria monocytogenes - genetics / Models, Molecular / multidisciplinary / Nuclease / Nucleic Acid Conformation / Phages / Protein Binding / Ribonucleic acid / RNA / RNA phages / RNA, Guide, CRISPR-Cas Systems - genetics / Science / Science (multidisciplinary) / Streptococcus pyogenes - genetics / Structure-Activity Relationship