한빛사 논문
Sungchul Kim1,5,*, Luuk Loeff1,4,5, Sabina Colombo1, Slobodan Jergic2,3, Stan J. J. Brouns1 & Chirlmin Joo1,*
1 Kavli Institute of Nanoscience, Department of Bionanoscience, Delft University of Technology, Delft, The Netherlands.
2 Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, New South Wales, Australia.
3 Illawarra Health and Medical Research Institute, Wollongong, New South Wales, Australia.
4 Present address: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
5 These authors contributed equally: Sungchul Kim, Luuk Loeff.
*Correspondence to Sungchul Kim or Chirlmin Joo
Abstract
CRISPR–Cas immunity protects prokaryotes against invading genetic elements 1. It uses the highly conserved Cas1–Cas2 complex to establish inheritable memory (spacers) 2,3,4,5. 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.
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