Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) systems have been effectively harnessed to engineer the genomes of organisms from across the tree of life. Nearly all currently characterized Cas9 proteins are derived from mesophilic bacteria, and canonical Cas9...
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| Format: | Article |
| Language: | English |
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Proceedings of the National Academy of Sciences
2020
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| Online Access: | https://hdl.handle.net/1721.1/124417 |
| _version_ | 1811083180717899776 |
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| author | Blainey, Paul C |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Blainey, Paul C |
| author_sort | Blainey, Paul C |
| collection | MIT |
| description | Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) systems have been effectively harnessed to engineer the genomes of organisms from across the tree of life. Nearly all currently characterized Cas9 proteins are derived from mesophilic bacteria, and canonical Cas9 systems are challenged by applications requiring enhanced stability or elevated temperatures. We discovered IgnaviCas9, a Cas9 protein from a hyperthermophilic Ignavibacterium identified through mini-metagenomic sequencing of samples from a hot spring. IgnaviCas9 is active at temperatures up to 100 °C in vitro, which enables DNA cleavage beyond the 44 °C limit of Streptococcus pyogenes Cas9 (SpyCas9) and the 70 °C limit of both Geobacillus stearothermophilus Cas9 (GeoCas9) and Geobacillus thermodenitrificans T12 Cas9 (ThermoCas9). As a potential application of this enzyme, we demonstrate that IgnaviCas9 can be used in bacterial RNA-seq library preparation to remove unwanted cDNA from 16s ribosomal rRNA without increasing the number of steps, thus underscoring the benefits provided by its exceptional thermostability in improving molecular biology and genomic workflows. IgnaviCas9 is an exciting addition to the CRISPR-Cas9 toolbox and expands its temperature range. |
| first_indexed | 2024-09-23T12:27:12Z |
| format | Article |
| id | mit-1721.1/124417 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T12:27:12Z |
| publishDate | 2020 |
| publisher | Proceedings of the National Academy of Sciences |
| record_format | dspace |
| spelling | mit-1721.1/1244172022-09-28T08:02:49Z Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium Blainey, Paul C Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Biological Engineering Multidisciplinary Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) systems have been effectively harnessed to engineer the genomes of organisms from across the tree of life. Nearly all currently characterized Cas9 proteins are derived from mesophilic bacteria, and canonical Cas9 systems are challenged by applications requiring enhanced stability or elevated temperatures. We discovered IgnaviCas9, a Cas9 protein from a hyperthermophilic Ignavibacterium identified through mini-metagenomic sequencing of samples from a hot spring. IgnaviCas9 is active at temperatures up to 100 °C in vitro, which enables DNA cleavage beyond the 44 °C limit of Streptococcus pyogenes Cas9 (SpyCas9) and the 70 °C limit of both Geobacillus stearothermophilus Cas9 (GeoCas9) and Geobacillus thermodenitrificans T12 Cas9 (ThermoCas9). As a potential application of this enzyme, we demonstrate that IgnaviCas9 can be used in bacterial RNA-seq library preparation to remove unwanted cDNA from 16s ribosomal rRNA without increasing the number of steps, thus underscoring the benefits provided by its exceptional thermostability in improving molecular biology and genomic workflows. IgnaviCas9 is an exciting addition to the CRISPR-Cas9 toolbox and expands its temperature range. 2020-03-30T18:47:40Z 2020-03-30T18:47:40Z 2019-10-28 2020-02-12T19:16:50Z Article http://purl.org/eprint/type/JournalArticle 0027-8424 1091-6490 https://hdl.handle.net/1721.1/124417 Schmidt, Stephanie Tzouanas et al. "Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium." Proceedings of the National Academy of Sciences of the United States of America 116(2019):23100-23105 © 2019 The Author(s) en 10.1073/pnas.1904273116 Proceedings of the National Academy of Sciences of the United States of America Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf Proceedings of the National Academy of Sciences PNAS |
| spellingShingle | Multidisciplinary Blainey, Paul C Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium |
| title | Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium |
| title_full | Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium |
| title_fullStr | Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium |
| title_full_unstemmed | Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium |
| title_short | Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium |
| title_sort | nucleic acid cleavage with a hyperthermophilic cas9 from an uncultured ignavibacterium |
| topic | Multidisciplinary |
| url | https://hdl.handle.net/1721.1/124417 |
| work_keys_str_mv | AT blaineypaulc nucleicacidcleavagewithahyperthermophiliccas9fromanunculturedignavibacterium |