Broad-spectrum enzymatic inhibition of CRISPR-Cas12a.
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AcrVA1 causes multiple-turnover destruction of the guide spacer in pre-existing Cas12a-crRNA complexes.
"AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the target-recognition sequence of the Cas12a-bound guide RNA to irreversibly inactivate the Cas12a complex."
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The cleavage is endoribonucleolytic, truncates the spacer, and permanently inactivates the complex
"we show that AcrVA1 triggers multiple turnover endoribonucleolytic cleavage of a Cas12a-bound crRNA to truncate the spacer sequence and permanently inactivate the complex"
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Scissile phosphates lie at positions five to eight of the crRNA spacer
"we mapped the scissile phosphates at positions five to eight within the crRNA spacer"
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Catalysis generates an intact 3-prime crRNA fragment that Cas12a then releases
"The activity is that of an endoribonuclease where catalysis generates an intact 3’-fragment of the crRNA that is released by Cas12a after AcrVA1-triggered truncation"
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Inhibition is sub-stoichiometric and therefore catalytic, which is the basis for separating enzymatic from stoichiometric anti-CRISPRs
"Thus, AcrVA1 activity is multiple-turnover where cleavage of a crRNA will permanently inactivate Cas12a-crRNA complexes through a mode of inhibition not previously observed for any anti-CRISPR protein."
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Inhibition of double-stranded DNA cleavage extends across three divergent Cas12a orthologues
"AcrVA1 blocked dsDNA cleavage by all three Cas12a orthologs"
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AcrVA1 also abolishes double-stranded DNA binding to Cas12a
"revealing that AcrVAs abolished dsDNA binding"
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AcrVA1 has no ribonuclease activity on crRNA in the absence of Cas12a
"AcrVA1 had no effect on the integrity of mature or pre-crRNA in the absence of Cas12a"
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The authors could not identify which component bears the catalytic centre, although they consider AcrVA1 the likely nuclease
"We demonstrated that the nuclease activity is entirely dependent on the presence of a Cas12a-crRNA complex and AcrVA1, but our data do not describe the identity of the component bearing the catalytic center for the observed nuclease activity. It is likely that AcrVA1 is an RNase, however we could not detect any RNase"