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RNA-guided DNA insertion with CRISPR-associated transposases.

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.

SUBMITTER: Strecker J

PROVIDER: S-EPMC6659118 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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RNA-guided DNA insertion with CRISPR-associated transposases.

Strecker Jonathan J Ladha Alim A Gardner Zachary Z Schmid-Burgk Jonathan L JL Makarova Kira S KS Koonin Eugene V EV Zhang Feng F

Science (New York, N.Y.) 20190606 6448

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 <i>Scytonema hofmanni</i> (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 pr ...[more]

PMID: 31171706

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