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

• Published in: eLife Vol. 8
• Main Authors: Forsberg, Kevin J, Bhatt, Ishan V, Schmidtke, Danica T, Javanmardi, Kamyab, Dillard, Kaylee E, Stoddard, Barry L, Finkelstein, Ilya J, Kaiser, Brett K, Malik, Harmit S
• Format: Journal Article
• Language: English
• Published: England eLife Science Publications, Ltd 10.09.2019; eLife Sciences Publications Ltd; eLife Sciences Publications, Ltd
• Subjects: anti-CRISPR; Antibiotics; Antiviral agents; Bacteria; bacterial defense systems; Bacterial genetics; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Biology; Cells (Biology); Chemical inhibitors; Cloning; CRISPR; CRISPR-Associated Protein 9 - antagonists & inhibitors; CRISPR-Cas; Deoxyribonucleic acid; DNA; Drug resistance; Ecosystems; Enzyme Inhibitors - isolation & purification; Enzyme Inhibitors - metabolism; Enzymes; Escherichia coli; Feces - microbiology; Feces - virology; functional metagenomics; Genes; Genetic aspects; Genetic research; Genetically modified organisms; Genomes; human microbiome; Humans; Identification and classification; Immune system; Medical research; Metagenomics; Microbial drug resistance; Microbiology and Infectious Disease; Microbiomes; Microbiota; Mouth - microbiology; Mouth - virology; Mutation; phage; Phages; Physiological aspects; Plasmids; Proteins; Transposons; Viral Proteins - genetics; Viral Proteins - isolation & purification; Viral Proteins - metabolism; United States; United States > US; anti-CRISPR; human microbiome; bacterial defense systems; infectious disease; functional metagenomics; CRISPR-Cas; microbiology; phage; S. pyogenes; human; E. coli
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.46540

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 Cas9 (SpyCas9) in . The most potent Acr from this set, AcrIIA11, was recovered from a 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 . 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.