Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor

Genome Biol. 2021 Jun 3;22(1):170. doi: 10.1186/s13059-021-02389-w.

Abstract

Prime editors, novel genome-editing tools consisting of a CRISPR-Cas9 nickase and an engineered reverse transcriptase, can induce targeted mutagenesis. Nevertheless, much effort is required to optimize and improve the efficiency of prime-editing. Herein, we introduce two strategies to improve the editing efficiency using proximal dead sgRNA and chromatin-modulating peptides. We used enhanced prime-editing to generate Igf2 mutant mice with editing frequencies of up to 47% and observed germline transmission, no off-target effects, and a dwarf phenotype. This improved prime-editing method can be efficiently applied to cell research and to generate mouse models.

Keywords: Adamts20; Chromatin-modulating peptides; Dwarf phenotype; Germline transmission; Igf2; Mouse cells and embryos; Prime editor; Proximal dead sgRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line
  • Cells / metabolism*
  • Chromatin / metabolism
  • Embryo, Mammalian / metabolism*
  • Gene Editing*
  • Humans
  • Mice, Inbred C57BL
  • Mice, Inbred ICR
  • Mutagenesis / genetics*
  • RNA, Guide / genetics

Substances

  • Chromatin
  • RNA, Guide