A catalogue of biochemically diverse CRISPR-Cas9 orthologs
| Autor: | Zhenglin Hou, Česlovas Venclovas, Peter Weigele, Megumu Mabuchi, Joshua K. Young, William E. Jack, Zhiyi Sun, Greta Bigelyte, Tautvydas Karvelis, Ezra Schildkraut, Virginijus Siksnys, N. Doane Chilcoat, Tomas Urbaitis, G. Brett Robb, Ryan T. Fuchs, Monika Jasnauskaite, Jennifer L. Curcuru, Mantvyda M. Grusyte, Shane K. Dooley, Po-Hao Wang, Giedrius Gasiunas, Clara M. Alarcon, Darius Kazlauskas, Mark Cigan, Sushmitha Paulraj |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Hydrolases Science General Physics and Astronomy Computational biology Article General Biochemistry Genetics and Molecular Biology Homology (biology) 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Genome editing CRISPR-Associated Protein 9 Sequence Homology Nucleic Acid CRISPR Clustered Regularly Interspaced Short Palindromic Repeats Guide RNA DNA Cleavage lcsh:Science Gene Editing Nuclease Genome Multidisciplinary Bacteria biology Base Sequence Cas9 Computational Biology General Chemistry Protospacer adjacent motif 030104 developmental biology chemistry nuclease biology.protein lcsh:Q CRISPR-Cas Systems 030217 neurology & neurosurgery DNA Biotechnology RNA Guide Kinetoplastida |
| Zdroj: | Nature communications, London : Nature Publishing Group, 2020, vol. 11, no. 1, art. no. 5512, p. [1-10] Nature Communications Nature Communications, Vol 11, Iss 1, Pp 1-10 (2020) |
| ISSN: | 2041-1723 |
| Popis: | Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases. A few bacterial Cas9 nucleases have been repurposed as genome editing tools. Here, the authors use bioinformatic and biochemical analyses to characterize 79 Cas9 proteins, revealing substantial functional diversity and thus expanding the available toolbox of RNA-programmable CRISPR-associated nucleases. |
| Databáze: | OpenAIRE |
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