Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome

Abstract

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 71Results 72 73 A functio...

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

“…However, creating 'designer' inhibitors for specific gene editors and editing scenarios is very challenging as Acr proteins are small and lack any sequence and structural conservation to use for direct identification of desired modalities. To overcome these problems, approaches such as screening 13 self-targeting genomes (7,10,19) or metagenomic DNA (13,14) attempt to cast a wide net in the genomic space to identify novel Acr activities. A more direct method is to use a 'guilt-byassociation' search approach, which leverages the repeated observations that Acrs tend to coexist in similar operons and that the genomic neighborhood of several Acrs contain anti-CRISPR associated (aca) genes that are closely coupled with Acrs, and widespread in bacteria and MGEs.…”

“…This motif is a prominent feature of aca genes, which were recently shown to negatively regulate the transcription of their own operon (16,34). The self-regulating activity of aca proteins was shown 14 to be mediated by a direct interaction between the HTH motif and promoter proximal sequences containing two inverted repeats (IR). Examining the promoter regions of AcrIIA13-15, we found that all of them also contain similar promoter-proximal IR sequences (Fig.…”

“…However, some MGEs, particularly phages, have evolved anti-CRISPRs (Acrs), peptide inhibitors of Cas proteins that block CRISPR defense systems (2,3). Acrs have been discovered to inhibit distinct CRISPR systems, including type I (4)(5)(6)(7)(8), type II (9)(10)(11)(12)(13)(14)(15)(16), type III (17,18), and type V (7,19). Strategies for identifying new Acrs include testing genes of unknown function that are proximal to anti-CRISPR associated (aca) genes (5-7, 9, 15) and screening genes in organisms with self-targeting CRISPR systems (7,19,20).…”

“…Multiple anti-CRISPR (Acr) families inhibit Streptococcus pyogenes Cas9 (SpyCas9) and various Cas12a proteins, and can be used in cell-based experiments to control genome editing outcomes (7,10,11,13,14,22,23). Although weak cross-reactivity with other non-cognate Cas9 orthologs has been detected for a subset of these, we wondered whether more potent and selective inhibitors of particular Cas9 variants might exist in nature.…”

Anti‐CRISPR proteins targeting the CRISPR‐Cas system enrich the toolkit for genetic engineering

“…Furthermore, compared with the previously identified Acrs, which were identified via sequence homology or a genetic context, AcrIIA7-10, which inhibits Cas9, were identified based on functional activity using a high-throughput approach from metagenomics libraries [57] (Table 1). Recently, Forsberg et al [58] independently developed a similar Acr search strategy to discover a newly potent Acr, named AcrIIA11, identified from a Lachnospiraceae phage and that inhibits spCas9 in bacteria and in human cells. Overall, this functional selection system offers a new strategy for discovering Acrs targeting desired Cas proteins, overcoming the limited availability of reference Acr families determined through computational discovery directed by genomic background or homology.…”