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

Kevin J Forsberg,Ishan V Bhatt,Barry L Stoddard,Kaylee E Dillard,Kamyab Javanmardi,Danica T Schmidtke,Harmit S Malik,Brett K Kaiser,Ilya J Finkelstein

doi:10.7554/elife.46540

Kevin J Forsberg, Ishan V Bhatt + Show 7 more

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https://doi.org/10.7554/elife.46540

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Abstract

CRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because Acrs are challenging to identify, their natural diversity and impact on microbial ecosystems are underappreciated. To overcome this discovery bottleneck, we developed a high-throughput functional selection to isolate ten DNA fragments from human oral and fecal metagenomes that inhibit Streptococcus pyogenes Cas9 (SpyCas9) in Escherichia coli. The most potent Acr from this set, AcrIIA11, was recovered from a Lachnospiraceae phage. We found that AcrIIA11 inhibits SpyCas9 in bacteria and in human cells. AcrIIA11 homologs are distributed across diverse bacteria; many distantly-related homologs inhibit both SpyCas9 and a divergent Cas9 from Treponema denticola. We find that AcrIIA11 antagonizes SpyCas9 using a different mechanism than other previously characterized Type II-A Acrs. Our study highlights the power of functional selection to uncover widespread Cas9 inhibitors within diverse microbiomes.

Highlights

CRISPR-Cas adaptive immune systems are present in many bacterial and archaeal genomes (Makarova et al, 2015; Burstein et al, 2016), where they protect their hosts from infection by phages (Barrangou et al, 2007), plasmids (Marraffini and Sontheimer, 2008), and other mobile genetic elements (MGEs) (Zhang et al, 2013)
We find that many unrelated metagenomic clones from human oral and gut microbiomes protect against Streptococcus pyogenes Cas9 (SpyCas9), the variant used most commonly for gene editing applications (Knott and Doudna, 2018)
Our selection interrogates core bacterial genomes as well as DNA from the phages, plasmids, and other mobile genetic elements that infect these bacteria, which must contend with CRISPR-Cas immunity

Summary

Introduction

CRISPR-Cas adaptive immune systems are present in many bacterial and archaeal genomes (Makarova et al, 2015; Burstein et al, 2016), where they protect their hosts from infection by phages (Barrangou et al, 2007), plasmids (Marraffini and Sontheimer, 2008), and other mobile genetic elements (MGEs) (Zhang et al, 2013). CRISPR-Cas systems mediate this defense by incorporating short (~30 bp) spacer sequences from invading genomes into an immunity locus in the host genome. These spacer sequences are expressed and processed into CRISPR RNAs (crRNAs) that, together with various Cas nucleases, mediate homology-dependent restriction of invading genomes. Phages and other MGEs have evolved dedicated CRISPR-Cas antagonists, called anti-CRISPRs (Acrs) (Bondy-Denomy et al, 2013), which can promote phage infection, enable horizontal gene transfer (HGT), and shape microbial ecosystems (Borges et al, 2017; Pawluk et al, 2017a). Acrs that inhibit the type II Cas (Pawluk et al, 2016a; Rauch et al, 2017)

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