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
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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