Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome
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 ove...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2019-09-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/46540 |
| _version_ | 1811235968097714176 |
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| author | Kevin J Forsberg Ishan V Bhatt Danica T Schmidtke Kamyab Javanmardi Kaylee E Dillard Barry L Stoddard Ilya J Finkelstein Brett K Kaiser Harmit S Malik |
| author_facet | Kevin J Forsberg Ishan V Bhatt Danica T Schmidtke Kamyab Javanmardi Kaylee E Dillard Barry L Stoddard Ilya J Finkelstein Brett K Kaiser Harmit S Malik |
| author_sort | Kevin J Forsberg |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T12:01:11Z |
| format | Article |
| id | doaj.art-475c27cc0e5d46fe8d33c6b81749e41f |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T12:01:11Z |
| publishDate | 2019-09-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-475c27cc0e5d46fe8d33c6b81749e41f2022-12-22T03:33:50ZengeLife Sciences Publications LtdeLife2050-084X2019-09-01810.7554/eLife.46540Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiomeKevin J Forsberg0https://orcid.org/0000-0002-1545-8925Ishan V Bhatt1Danica T Schmidtke2Kamyab Javanmardi3Kaylee E Dillard4Barry L Stoddard5Ilya J Finkelstein6https://orcid.org/0000-0002-9371-2431Brett K Kaiser7Harmit S Malik8https://orcid.org/0000-0001-6005-0016Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United StatesDivision of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United StatesDivision of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United StatesDepartment of Molecular Biosciences and Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, United StatesDepartment of Molecular Biosciences and Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, United StatesDivision of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United StatesDepartment of Molecular Biosciences and Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, United States; Center for Systems Biology and Synthetic Biology, University of Texas at Austin, Austin, United StatesDivision of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States; Department of Biology, Seattle University, Seattle, United StatesDivision of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States; Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, United StatesCRISPR-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.https://elifesciences.org/articles/46540CRISPR-Casanti-CRISPRfunctional metagenomicshuman microbiomephagebacterial defense systems |
| spellingShingle | Kevin J Forsberg Ishan V Bhatt Danica T Schmidtke Kamyab Javanmardi Kaylee E Dillard Barry L Stoddard Ilya J Finkelstein Brett K Kaiser Harmit S Malik Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome eLife CRISPR-Cas anti-CRISPR functional metagenomics human microbiome phage bacterial defense systems |
| title | Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome |
| title_full | Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome |
| title_fullStr | Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome |
| title_full_unstemmed | Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome |
| title_short | Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome |
| title_sort | functional metagenomics guided discovery of potent cas9 inhibitors in the human microbiome |
| topic | CRISPR-Cas anti-CRISPR functional metagenomics human microbiome phage bacterial defense systems |
| url | https://elifesciences.org/articles/46540 |
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