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

Jakhanwal, Shrutee; Cress, Brady F.; Maguin, Pascal; Lobba, Marco J.; Marraffini, Luciano A.; Doudna, Jennifer A.

doi:10.1093/nar/gkab123

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

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Abstract

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.

Authors:

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

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.
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.
Rockefeller Univ., New York, NY (United States). Lab. of Bacteriology
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
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)

Publication Date:: Mon Mar 08 00:00:00 EST 2021

Research Org.:: Univ. of California, Berkeley, CA (United States)

Sponsoring Org.:: USDOE Office of Science (SC); National Institutes of Health (NIH); National Science Foundation (NSF); Defense Advanced Research Projects Agency (DARPA)

OSTI Identifier:: 1815949

Grant/Contract Number:: AC02-05CH11231; U24HG010423; 1817593; HR0011-17-2-0043; N660012024033; RM1HG009490; F32 GM131654

Resource Type:: Accepted Manuscript

Journal Name:: Nucleic Acids Research

Additional Journal Information:: Journal Volume: 49; Journal Issue: 6; Journal ID: ISSN 0305-1048

Publisher:: Oxford University Press

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; Nucleic Acid Enzymology; Genomics

Citation Formats


                    Jakhanwal, Shrutee, Cress, Brady F., Maguin, Pascal, Lobba, Marco J., Marraffini, Luciano A., and Doudna, Jennifer A. A CRISPR-Cas9–integrase complex generates precise DNA fragments for genome integration.  United States: N. p., 2021. 
Web.  doi:10.1093/nar/gkab123.

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                    Jakhanwal, Shrutee, Cress, Brady F., Maguin, Pascal, Lobba, Marco J., Marraffini, Luciano A., & Doudna, Jennifer A. A CRISPR-Cas9–integrase complex generates precise DNA fragments for genome integration.  United States.  https://doi.org/10.1093/nar/gkab123

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                    Jakhanwal, Shrutee, Cress, Brady F., Maguin, Pascal, Lobba, Marco J., Marraffini, Luciano A., and Doudna, Jennifer A. Mon .  
"A CRISPR-Cas9–integrase complex generates precise DNA fragments for genome integration".  United States.  https://doi.org/10.1093/nar/gkab123.  https://www.osti.gov/servlets/purl/1815949.

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@article{osti_1815949,

  title        = {A CRISPR-Cas9–integrase complex generates precise DNA fragments for genome integration},

  author       = {Jakhanwal, Shrutee and Cress, Brady F. and Maguin, Pascal and Lobba, Marco J. and Marraffini, Luciano A. and Doudna, Jennifer A.},

  abstractNote = {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.},

  doi          = {10.1093/nar/gkab123},

  journal      = {Nucleic Acids Research},

  number       = 6,

  volume       = 49,

  place        = {United States},

  year         = {Mon Mar 08 00:00:00 EST 2021},

  month        = {Mon Mar 08 00:00:00 EST 2021}

}

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