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Cell. 2015 Nov 5;163(4):840-53. doi: 10.1016/j.cell.2015.10.008. Epub 2015 Oct 17.

Structural and Mechanistic Basis of PAM-Dependent Spacer Acquisition in CRISPR-Cas Systems.

Author information

1
Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
2
Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
3
Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: ylwang@ibp.ac.cn.

Abstract

Bacteria acquire memory of viral invaders by incorporating invasive DNA sequence elements into the host CRISPR locus, generating a new spacer within the CRISPR array. We report on the structures of Cas1-Cas2-dual-forked DNA complexes in an effort toward understanding how the protospacer is sampled prior to insertion into the CRISPR locus. Our study reveals a protospacer DNA comprising a 23-bp duplex bracketed by tyrosine residues, together with anchored flanking 3' overhang segments. The PAM-complementary sequence in the 3' overhang is recognized by the Cas1a catalytic subunits in a base-specific manner, and subsequent cleavage at positions 5 nt from the duplex boundary generates a 33-nt DNA intermediate that is incorporated into the CRISPR array via a cut-and-paste mechanism. Upon protospacer binding, Cas1-Cas2 undergoes a significant conformational change, generating a flat surface conducive to proper protospacer recognition. Here, our study provides important structure-based mechanistic insights into PAM-dependent spacer acquisition.

PMID:
26478180
DOI:
10.1016/j.cell.2015.10.008
[Indexed for MEDLINE]
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