Disabling Cas9 by an anti-CRISPR DNA mimic
Models, Molecular
0301 basic medicine
570
1.1 Normal biological development and functioning
Molecular Conformation
Cell Line
Structure-Activity Relationship
03 medical and health sciences
Models
CRISPR-Associated Protein 9
Genetics
2.1 Biological and endogenous factors
Humans
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Silencing
Research Articles
Gene Editing
Molecular
High-Throughput Nucleotide Sequencing
DNA
Biological Sciences
3. Good health
5.1 Pharmaceuticals
RNA
Biochemistry and Cell Biology
Generic health relevance
CRISPR-Cas Systems
Guide
Biotechnology
RNA, Guide, Kinetoplastida
DOI:
10.1126/sciadv.1701620
Publication Date:
2017-07-13T01:00:34Z
AUTHORS (10)
ABSTRACT
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated human cells. However, mechanism inhibition not known, potential applications for Cas9 inhibitor proteins mammalian cells have been fully established. We that anti-CRISPR protein AcrIIA4 binds only assembled Cas9-single-guide RNA (sgRNA) complexes alone. A 3.9 Å resolution cryo-electron microscopy structure Cas9-sgRNA-AcrIIA4 complex revealed surface highly acidic with 1:1 stoichiometry region normally engages DNA protospacer adjacent motif. Consistent this binding mode, order-of-addition experiments showed interferes recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery into as either or expression plasmid allows on-target while reducing off-target edits. These results provide mechanistic understanding function demonstrate can modulate extent outcomes editing.
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