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Mol Cell. 2019 Jan 8. pii: S1097-2765(18)31069-4. doi: 10.1016/j.molcel.2018.12.015. [Epub ahead of print]

A Functional Mini-Integrase in a Two-Protein-type V-C CRISPR System.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
2
Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
3
California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA 94720, USA.
4
Department of Energy, Joint Genome Institute, Walnut Creek, CA 94598, USA.
5
Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA 94720, USA.
6
Department of Earth and Planetary Sciences, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Environmental Science, Policy, and Management, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
7
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Gladstone Institutes, San Francisco, CA 94158, USA. Electronic address: doudna@berkeley.edu.

Abstract

CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a mini-integrase comprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17- to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.

KEYWORDS:

CRISPR; integrase; protein-DNA recognition; spacer acquisition

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