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Science. 2019 Jul 5;365(6448):48-53. doi: 10.1126/science.aax9181. Epub 2019 Jun 6.

RNA-guided DNA insertion with CRISPR-associated transposases.

Strecker J1,2,3,4, Ladha A1,2,3,4, Gardner Z1,2,3,4, Schmid-Burgk JL1,2,3,4, Makarova KS5, Koonin EV5, Zhang F6,2,3,4.

Author information

1
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
2
McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
3
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
4
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
5
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
6
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. zhang@broadinstitute.org.

Abstract

CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the Escherichia coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion.

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PMID:
31171706
PMCID:
PMC6659118
[Available on 2020-01-05]
DOI:
10.1126/science.aax9181

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