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Selective loading and processing of prespacers for precise CRISPR adaptation

Journal Article


Abstract


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

Authors


  •   Kim, Sungchul (external author)
  •   Loeff, Luuk (external author)
  •   Colombo, Sabina (external author)
  •   Jergic, Slobodan
  •   Brouns, Stan (external author)
  •   Joo, Chirlmin (external author)

Publication Date


  • 2020

Published In


Citation


  • Kim, S., Loeff, L., Colombo, S., Jergic, S., Brouns, S. & Joo, C. (2020). Selective loading and processing of prespacers for precise CRISPR adaptation. Nature,

Scopus Eid


  • 2-s2.0-85079775402

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers1/1244

Has Global Citation Frequency


Place Of Publication


  • United Kingdom

Abstract


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

Authors


  •   Kim, Sungchul (external author)
  •   Loeff, Luuk (external author)
  •   Colombo, Sabina (external author)
  •   Jergic, Slobodan
  •   Brouns, Stan (external author)
  •   Joo, Chirlmin (external author)

Publication Date


  • 2020

Published In


Citation


  • Kim, S., Loeff, L., Colombo, S., Jergic, S., Brouns, S. & Joo, C. (2020). Selective loading and processing of prespacers for precise CRISPR adaptation. Nature,

Scopus Eid


  • 2-s2.0-85079775402

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers1/1244

Has Global Citation Frequency


Place Of Publication


  • United Kingdom