Extended Data Fig. 3: AcrVA2 does not inhibit or downregulate cas12a in vitro.
From: Translation-dependent degradation of cas12 mRNA triggered by an anti-CRISPR
(a) In vitro cleavage assay with purified anti-CRISPR protein added to MbCas12a(33362) ribonucleoprotein complexes programmed to target a double-stranded DNA amplicon. Cas12a and crRNA were assembled into ribonucleoprotein complexes before addition of anti-CRISPR or control. Anti-CRISPR or control protein was added at a 1:10 or 1:1 ratio relative to Cas12a. (b) In vitro cleavage assay with purified anti-CRISPR protein added to apoMbCas12a(33362) before addition of crRNA and target dsDNA. Anti-CRISPR or control protein was added in a 10:1 or 1:1 ratio relative to apoCas12a. (c) Phage plaque assay using ten-fold serial dilution of phage to assess Cas12a inhibition by 10xHis-MBP-AcrVA2 relative to AcrVA2 and vector control. (d) Phage plaque assay on strains expressing different homologs of mbcas12a from M. bovoculi strains 237 and 33362. Ten-fold serial dilutions of phage were plated on bacterial lawns to assess Cas12a inhibition. (e) Western blot on bacterial lysates to assess downregulation of different mbcas12 orthologs by AcrVA2 E98A/D129A/D195A relative to controls. Experiments in (a–e) were independently repeated at least twice with similar results.
