A CRISPR-Cas9–integrase complex generates precise DNA fragments for genome integration

Journal Article · Mon Mar 08 00:00:00 EST 2021 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkab123· OSTI ID:1815949

Jakhanwal, Shrutee ^[1]; Cress, Brady F. ^[2]; Maguin, Pascal ^[3]; Lobba, Marco J. ^[4]; Marraffini, Luciano A. ^[3]; Doudna, Jennifer A. ^[5]

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1. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). California Institute for Quantitative Biosciences; Univ. of California, Berkeley, CA (United States). Innovative Genomics Inst.; OSTI
2. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). California Institute for Quantitative Biosciences; Univ. of California, Berkeley, CA (United States). Innovative Genomics Inst.
3. Rockefeller Univ., New York, NY (United States). Lab. of Bacteriology
4. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). California Institute for Quantitative Biosciences; Univ. of California, Berkeley, CA (United States). Dept. of Chemistry
5. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology. California Inst. for Quantitative Biosciences. Innovative Genomics Inst. Dept. of Chemistry. Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division; Gladstone Inst. of Data Science and Biotechnology, San Francisco, CA (United States)

CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9 s activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes.

• Research Organization: Univ. of California, Berkeley, CA (United States)
• Sponsoring Organization: Defense Advanced Research Projects Agency (DARPA); National Institutes of Health (NIH); National Science Foundation (NSF); USDOE Office of Science (SC)
• Grant/Contract Number: AC02-05CH11231
• OSTI ID: 1815949
• Journal Information: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 6 Vol. 49; ISSN 0305-1048
• Publisher: Oxford University Press
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Genomics
Nucleic Acid Enzymology