Functional selection reveals a metagenomic contig encoding a novel SpyCas9 inhibitor.
(A) A plasmid protection assay was used to reveal SpyCas9 inhibition. In this assay, plasmids without SpyCas9 inhibitors are cleaved by Cas9 and do not give rise to KanR colonies, whereas those encoding inhibitors withstand SpyCas9 attack and yield KanR colonies. (B) The contig F01A_4 protects a plasmid from SpyCas9 attack, but an early stop codon in orf_1 (Δ1) eliminates this phenotype. Stop codons in orf_2 or orf_3 (Δ2 and Δ3) have no effect. Thus, we conclude that orf_1 is necessary for inhibition of SpyCas9. Asterisks depict statistically significant differences in plasmid retention between the indicated genotype and an empty vector control in SpyCas9-inducing conditions (Student t test, p < 0.002, n = 3); ns indicates no significance. All p-values were corrected for multiple hypotheses using Bonferroni method. (C) Expression of orf_1 (which we name acrIIA22) is sufficient for SpyCas9 antagonism, protecting a plasmid as effectively as acrIIA4. Asterisks are as in panel B but relate to the GFP negative control rather than to an empty vector. The individual numerical values that underlie the summary data in this figure may be found in S1 Data. KanR, kanamycin resistance; SpyCas9, Streptococcus pyogenes Cas9.