RNA-guided DNA insertion with CRISPR-associated transposases

By Jonathan Strecker, Alim Ladha, Zachary Gardner, Jonathan L. Schmid-Burgk, Kira S. Makarova, Eugene V. Koonin, Feng Zhang. Science, 2019

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Beyond adaptive immunity Prokaryotic CRISPR-Cas systems defend bacterial cells from phage and plasmid infection. Strecker et al. characterized a CRISPR-Cas system that functions beyond adaptive immunity (see the Perspective by Hou and Zhang). Type V-K CRISPR-Cas from cyanobacteria was associated with a Tn7-like transposon and a natural nuclease–deficient effector Cas12k. Cas12k directed the insertion of Tn7-like transposons into target sites via RNA-guided Tn7 transposition. This system was reprogrammed to efficiently and specifically insert DNA both in vitro and into the Escherichia coli genome. Science, this issue p. 48; see also p. 25 RNA-guided CRISPR-associated transposase is reprogrammed to achieve efficient and specific DNA insertion into the E. coli genome. 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.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/b3bcca7197e660e3855ea1292e6c79e62447f06f

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