Bacterial Retrons Enable Precise Gene Editing in Human Cells

CRISPR J. 2022 Feb;5(1):31-39. doi: 10.1089/crispr.2021.0065. Epub 2022 Jan 24.

Abstract

Retrons are bacterial genetic elements involved in anti-phage defense. They have the unique ability to reverse transcribe RNA into multicopy single-stranded DNA (msDNA) that remains covalently linked to their template RNA. Retrons coupled with CRISPR-Cas9 in yeast have been shown to improve the efficiency of precise genome editing via homology-directed repair (HDR). In human cells, HDR editing efficiency has been limited by challenges associated with delivering extracellular donor DNA encoding the desired mutation. In this study, we tested the ability of retrons to produce msDNA as donor DNA and facilitate HDR by tethering msDNA to guide RNA in HEK293T and K562 cells. Through heterologous reconstitution of retrons from multiple bacterial species with the CRISPR-Cas9 system, we demonstrated HDR rates of up to 11.4%. Overall, our findings represent the first step in extending retron-based precise gene editing to human cells.

Publication types

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

MeSH terms

  • CRISPR-Cas Systems / genetics
  • DNA, Bacterial / genetics
  • Gene Editing*
  • HEK293 Cells
  • Humans
  • RNA-Directed DNA Polymerase* / genetics
  • RNA-Directed DNA Polymerase* / metabolism

Substances

  • DNA, Bacterial
  • RNA-Directed DNA Polymerase