Selective loading and processing of prespacers for precise CRISPR adaptation

Sungchul Kim; Luuk Loeff; Sabina Colombo; Slobodan Jergic; Stan J.J. Brouns; Chirlmin Joo

doi:10.1038/s41586-020-2018-1

Selective loading and processing of prespacers for precise CRISPR adaptation

Sungchul Kim, Luuk Loeff, Sabina Colombo, Slobodan Jergic, Stan J.J. Brouns, Chirlmin Joo

Department of Physics

Research output: Contribution to journal › Article › peer-review

39 Scopus citations

Abstract

CRISPR–Cas immunity protects prokaryotes against invading genetic elements¹. It uses the highly conserved Cas1–Cas2 complex to establish inheritable memory (spacers)^2–5. How Cas1–Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear^6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1–Cas2 selects precursors of prespacers from DNA in various forms—including single-stranded DNA and partial duplexes—in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1–Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1–Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.

Original language	English
Pages (from-to)	141-145
Number of pages	5
Journal	Nature
Volume	579
Issue number	7797
DOIs	https://doi.org/10.1038/s41586-020-2018-1
State	Published - 5 Mar 2020

Bibliographical note

Funding Information:
Acknowledgements We thank A. C. Haagsma and T. Künne for providing Cas1–Cas2 vectors and proteins; S. Leachman, N. Dekker and the members of the C.J. and S.J.J.B. laboratories for discussions; and T. J. Cui for discussions on kinetic models. S.J. thanks N. Dixon for guidance. S.K. was partly funded by a Marie Skłodowska-Curie grant (753528); C.J. and S.J.J.B. were funded by the Foundation for Fundamental Research on Matter (15PR3188); and S.J. was funded by a collaborative grant from King Abdullah University of Science and Technology, Saudi Arabia (OSR-2015-CRG4-2644).

Publisher Copyright:
© 2020, The Author(s), under exclusive licence to Springer Nature Limited.

Access to Document

10.1038/s41586-020-2018-1

Cite this

@article{ec8a42ceeb044abb9e5bd9f32daece76,

title = "Selective loading and processing of prespacers for precise CRISPR adaptation",

abstract = "CRISPR–Cas immunity protects prokaryotes against invading genetic elements1. It uses the highly conserved Cas1–Cas2 complex to establish inheritable memory (spacers)2–5. How Cas1–Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1–Cas2 selects precursors of prespacers from DNA in various forms—including single-stranded DNA and partial duplexes—in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1–Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1–Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.",

author = "Sungchul Kim and Luuk Loeff and Sabina Colombo and Slobodan Jergic and Brouns, {Stan J.J.} and Chirlmin Joo",

note = "Funding Information: Acknowledgements We thank A. C. Haagsma and T. K{\"u}nne for providing Cas1–Cas2 vectors and proteins; S. Leachman, N. Dekker and the members of the C.J. and S.J.J.B. laboratories for discussions; and T. J. Cui for discussions on kinetic models. S.J. thanks N. Dixon for guidance. S.K. was partly funded by a Marie Sk{\l}odowska-Curie grant (753528); C.J. and S.J.J.B. were funded by the Foundation for Fundamental Research on Matter (15PR3188); and S.J. was funded by a collaborative grant from King Abdullah University of Science and Technology, Saudi Arabia (OSR-2015-CRG4-2644). Publisher Copyright: {\textcopyright} 2020, The Author(s), under exclusive licence to Springer Nature Limited.",

year = "2020",

month = mar,

day = "5",

doi = "10.1038/s41586-020-2018-1",

language = "English",

volume = "579",

pages = "141--145",

journal = "Nature",

issn = "0028-0836",

publisher = "Nature Research",

number = "7797",

}

TY - JOUR

T1 - Selective loading and processing of prespacers for precise CRISPR adaptation

AU - Kim, Sungchul

AU - Loeff, Luuk

AU - Colombo, Sabina

AU - Jergic, Slobodan

AU - Brouns, Stan J.J.

AU - Joo, Chirlmin

N1 - Funding Information: Acknowledgements We thank A. C. Haagsma and T. Künne for providing Cas1–Cas2 vectors and proteins; S. Leachman, N. Dekker and the members of the C.J. and S.J.J.B. laboratories for discussions; and T. J. Cui for discussions on kinetic models. S.J. thanks N. Dixon for guidance. S.K. was partly funded by a Marie Skłodowska-Curie grant (753528); C.J. and S.J.J.B. were funded by the Foundation for Fundamental Research on Matter (15PR3188); and S.J. was funded by a collaborative grant from King Abdullah University of Science and Technology, Saudi Arabia (OSR-2015-CRG4-2644). Publisher Copyright: © 2020, The Author(s), under exclusive licence to Springer Nature Limited.

PY - 2020/3/5

Y1 - 2020/3/5

N2 - CRISPR–Cas immunity protects prokaryotes against invading genetic elements1. It uses the highly conserved Cas1–Cas2 complex to establish inheritable memory (spacers)2–5. How Cas1–Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1–Cas2 selects precursors of prespacers from DNA in various forms—including single-stranded DNA and partial duplexes—in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1–Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1–Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.

AB - CRISPR–Cas immunity protects prokaryotes against invading genetic elements1. It uses the highly conserved Cas1–Cas2 complex to establish inheritable memory (spacers)2–5. How Cas1–Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1–Cas2 selects precursors of prespacers from DNA in various forms—including single-stranded DNA and partial duplexes—in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1–Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1–Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.

UR - http://www.scopus.com/inward/record.url?scp=85079775402&partnerID=8YFLogxK

U2 - 10.1038/s41586-020-2018-1

DO - 10.1038/s41586-020-2018-1

M3 - Article

C2 - 32076262

AN - SCOPUS:85079775402

SN - 0028-0836

VL - 579

SP - 141

EP - 145

JO - Nature

JF - Nature

IS - 7797

ER -

Selective loading and processing of prespacers for precise CRISPR adaptation

Abstract

Bibliographical note

Access to Document

Fingerprint

Cite this