Abstract
CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly.
Highlights
CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements
When a bacterial cell is invaded by a phage, the CRISPR-Cas system acquires a short segment of the phage genome and integrates it into the CRISPR locus where it can serve as a template for the production of mature CRISPR RNA molecules
We previously showed that anti-CRISPR protein AcrIIC2Nme was able to robustly inhibit the cleavage activity of the N. meningitidis type II-C CRISPRCas[9] protein[9,23]
Summary
CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The transition of Cas[9] to its active conformation requires binding of a guide RNA molecule[25,26] This results in substantial structural rearrangements, with the most prominent conformational changes taking place in the REC lobe[27]. Binding to both target DNA and guide RNA are thought to be key regulators of Cas[9] enzyme function[26]. We show that AcrIIC2Nme functions by inhibiting loading of the guide RNA molecule, thereby preventing formation of the active CRISPR-Cas[9] surveillance complex. As previously characterized mechanisms of anti-CRISPR activity all target fully assembled CRISPR-Cas complexes, AcrIIC2Nme provides a unique mechanism for antiCRISPR activity
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