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Science. 2019 Jun 6. pii: eaax9181. doi: 10.1126/science.aax9181. [Epub ahead of print]

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 to manipulate nucleic acids; however, targeted insertion of DNA remains a challenge as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase (CAST) from cyanobacteria Scytonema hofmanni which 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 60-66 bp downstream of the protospacer. ShCAST integrates DNA into unique sites in the E. 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.

PMID:
31171706
DOI:
10.1126/science.aax9181

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