Several AcrIIA22 homologs in the CAG-217 clostridial genus can inhibit SpyCas9.
(A) A phylogeny of all unique AcrIIA22 homologs identified from metagenomic and NCBI databases. Phylogenetic classifications were assigned corresponding to the GTDB naming convention (Methods). Prophage sequences are shaded brown and homologs from hypervariable bacterial genomic islands are shaded yellow. Sequences obtained from NCBI are labeled with protein accession numbers. In other cases, AcrIIA22 homologs are numbered to match their contig-of-origin (S3 Table). In some cases, more than one AcrIIA22 homolog is found on the same contig (“gi” or “p” indicates its presence in a hypervariable genomic island or prophage genome, respectively). Circles at nodes indicate bootstrap support ≥ 0.75. Dashed boxes separate sequences identified from CAG-217 versus Eubacterium_R bacterial genera. Filled green circles indicate homologs that were tested for their ability to inhibit SpyCas9 in the plasmid protection assay in panel B. These homologs have been named with “a,” “b,” or “c” suffixes to distinguish them from the original AcrIIA22 metagenomic hit; their amino acid identity to the original hit is shown in parentheses. (B) Several homologs of AcrIIA22 in CAG-217 genomes inhibit SpyCas9. Asterisks depict statistically significant differences in plasmid retention under SpyCas9-inducing conditions between the indicated sample and a null mutant with an early stop codon in acrIIA22, as indicated in the legend at right (ns indicates no significance; p > 0.05). All p-values were corrected for multiple hypotheses using Bonferroni method (Student t test, n = 3). (C) AcrIIA22 inhibits divergent Cas9 proteins from Streptococcus pyogenes (SpyCas9) or Neisseria meningitidis (NmCas9) but not Cas12 proteins from Lachnospiraceae bacterium (LbCas12) or Francisella novicida (FnCas12). As in panel B, green bars indicate samples with expression of the indicated Cas nuclease, while unexpressed controls are depicted with gray lines. For Cas-expressing samples, significance was determined via a Student t test (n = 3) and denoted as follows: “*,” p ≤ 0.05; “**” p ≤ 0.001; “ns” no significance. Due to slight differences in the plasmid protection assay in panel C compared to panel B, A22 was retested against SpyCas9 to confirm activity (Methods). The individual numerical values that underlie the summary data in this figure may be found in S1 Data. GTDB, genome taxonomy database; SpyCas9, Streptococcus pyogenes Cas9.