RNA-guided DNA insertion with CRISPR-associated transposases

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics
  • CRISPR-Cas Systems / physiology*
  • Cyanobacteria / enzymology*
  • Cyanobacteria / genetics
  • DNA Transposable Elements*
  • Gene Editing / methods*
  • Mutagenesis, Insertional*
  • RNA, Guide*
  • Transposases / chemistry*
  • Transposases / genetics
  • Transposases / isolation & purification

Substances

  • DNA Transposable Elements
  • RNA, Guide
  • Transposases

Supplementary concepts

  • Scytonema hofmannii