Impaired nicking activity of AcrIIA22 variants in vitro correlates with lower SpyCas9 inhibition in vivo.
(A) Alanine mutagenesis of acidic amino acid residues (glutamic acid or aspartic acid) in AcrIIA22 reveals that D14 is important for plasmid protection against SpyCas9. Asterisks depict statistically significant differences in plasmid retention under SpyCas9-inducing and noninducing conditions, per the legend at right. The D14A mutant is significantly impaired, the E4A mutant is slightly impaired, whereas all other mutants are not impaired for plasmid protection against SpyCas9 compared to an uninduced control. All p-values were corrected for multiple hypotheses using Bonferroni method (Student t test, n = 3). (B) AcrIIA22 (black), AcrIIA22a (dark gray), and a D14A mutant (light gray) all elute with similar oligomer profiles via SEC. The dashed trace depicts protein standards of the indicated molecular weight, in kDa. (C) AcrIIA22a and the D14A mutant are impaired for nicking relative to AcrIIA22. All experiments were performed in triplicate, with standard deviations indicated by dashed lines (in most cases, the data points obscure these error bars). Asterisks denote cases where AcrIIA22 is significantly different than both AcrIIA22a and the D14A mutant after correcting for multiple hypotheses (Student t test, n = 3, Bonferroni correction). A single asterisk (*) means that adjusted p-values for both comparisons are below 0.05. A double asterisk (**) means that adjusted p-values are both below 0.005. S10G and S10H Fig show representative gels for these nicking experiments. The individual numerical values that underlie the summary data in this figure may be found in S1 Data. CFU, colony-forming unit; SEC, size exclusion chromatography; SpyCas9, Streptococcus pyogenes Cas9.