An Engineered Cas-Transposon System for Programmable and Site-Directed DNA Transpositions

CRISPR J. 2019 Dec;2(6):376-394. doi: 10.1089/crispr.2019.0030. Epub 2019 Nov 19.

Abstract

Efficient site-directed insertion of heterologous DNA into a genome remains an outstanding challenge. Recombinases that can integrate kilobase-sized DNA constructs are difficult to reprogram to user-defined loci, while genomic insertion using CRISPR-Cas methods relies on inefficient host DNA repair machinery. Here, we describe a Cas-Transposon (CasTn) system for genomic insertions that uses a Himar1 transposase fused to a catalytically dead dCas9 nuclease to mediate programmable, site-directed transposition. Using cell-free in vitro assays, we demonstrated that the Himar-dCas9 fusion protein increased the frequency of transposon insertion at a single targeted TA dinucleotide by >300-fold compared to a random transposase, and that site-directed transposition is dependent on target choice while robust to log-fold variations in protein and DNA concentrations. We also showed that Himar-dCas9 mediates directed transposition into plasmids in Escherichia coli. This work highlights CasTn as a new modality for host-independent, programmable, site-directed DNA insertions.

Publication types

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

MeSH terms

  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems / genetics
  • CRISPR-Cas Systems / physiology
  • DNA Transposable Elements / genetics*
  • DNA Transposable Elements / physiology
  • Endonucleases / genetics
  • Escherichia coli / genetics
  • Gene Editing / methods
  • Mutagenesis, Insertional / methods*
  • Recombination, Genetic / genetics
  • Recombination, Genetic / physiology
  • Transposases / genetics
  • Transposases / metabolism*

Substances

  • DNA Transposable Elements
  • Transposases
  • CRISPR-Associated Protein 9
  • Endonucleases