Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Proceedings of the National Academy of Sciences of the United States of America
Kyle E. WattersJennifer Doudna

Abstract

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structu...Continue Reading

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Aug 21, 2020·Human Gene Therapy·Amber Vu, Paul B McCray
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Methods Mentioned

BETA
electrophoretic mobility shift
flow cytometry
PCR
PCRs
ion exchange chromatography
size exclusion chromatography
fluorescence microscopy
transfection
transgenic

Software Mentioned

- Target Spacer Searcher ( STSS )
MUSCLE
blastp
Islander
Addgene
Self
HHpred
PHASTER

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