1 CRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, 2 which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because they are 3 difficult to identify, the natural diversity and impact of Acrs on microbial ecosystems is underappreciated. 4To overcome this discovery bottleneck, we developed a high-throughput functional selection that isolates 5 acr genes based on their ability to inhibit CRISPR-Cas function. Using this selection, we discovered ten 6 DNA fragments from human oral and fecal metagenomes that antagonize Streptococcus pyogenes Cas9 7 (SpyCas9). The most potent acr discovered, acrIIA11, was recovered from a Lachnospiraceae phage 8 and is among the strongest known SpyCas9 inhibitors. AcrIIA11 homologs are distributed across multiple 9 bacterial phyla and many divergent homologs inhibit SpyCas9. We show that AcrIIA11 antagonizes 10 SpyCas9 using a different mechanism than that of previously characterized inhibitors. Our study 11 highlights the power of functional selections to uncover widespread Cas9 inhibitors within diverse 12 microbiomes. 13 4 an Acr protein Watters et al., 2018). These clever strategies have revealed many 49 candidate acrs. But they have also highlighted the difficulty of finding new acr genes based on homology, 50 since acrs share little sequence conservation (Sontheimer and Davidson, 2017). As a result, most acrs 51 almost certainly lie unrecognized among the many genes of unknown function in phages, plasmids, and 52 other MGEs (Hatfull, 2015). 53
54To overcome the challenges associated with anti-CRISPR discovery, we devised a functional 55 metagenomic selection that identifies acr genes from any cloned DNA, based on their ability to protect a 56 plasmid from CRISPR-Cas-mediated destruction. Because functional metagenomics selects for a 57 function of interest from large clone libraries (Handelsman, 2004), it is well-suited to identify individual 58 genes like acrs that have strong fitness impacts (Iqbal et al., 2014;Forsberg et al., 2015; Forsberg et al., 59 2016; Genee et al., 2016). This approach may be particularly useful for Acr discovery because Acrs are 60 expressed from single genes and function readily in many genetic backgrounds (Pawluk et al., 2016a; 61 Rauch et al., 2017). 62
63Using this functional selection, we find that many unrelated metagenomic clones from human oral 64 and gut microbiomes protect against Streptococcus pyogenes Cas9 (SpyCas9), the variant used most 65 commonly for gene editing applications (Knott and Doudna, 2018). We identify a broadly distributed but 66 previously undescribed Acr from the most potent Cas9-antagonizing clone in our libraries. This Acr, 67 named AcrIIA11, binds both SpyCas9 and double-stranded DNA (dsDNA) and exhibits a novel mode of 68 SpyCas9 antagonism, protecting both plasmids and phages from immune restriction. Thus, our functional 69 approach reveals not only new classes of Acrs, but also new mechanisms of CRISPR-Cas antagonism. 70
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