Deletion and replacement of long genomic sequences using prime editing

Nat Biotechnol. 2022 Feb;40(2):227-234. doi: 10.1038/s41587-021-01026-y. Epub 2021 Oct 14.


Genomic insertions, duplications and insertion/deletions (indels), which account for ~14% of human pathogenic mutations, cannot be accurately or efficiently corrected by current gene-editing methods, especially those that involve larger alterations (>100 base pairs (bp)). Here, we optimize prime editing (PE) tools for creating precise genomic deletions and direct the replacement of a genomic fragment ranging from ~1 kilobases (kb) to ~10 kb with a desired sequence (up to 60 bp) in the absence of an exogenous DNA template. By conjugating Cas9 nuclease to reverse transcriptase (PE-Cas9) and combining it with two PE guide RNAs (pegRNAs) targeting complementary DNA strands, we achieve precise and specific deletion and repair of target sequences via using this PE-Cas9-based deletion and repair (PEDAR) method. PEDAR outperformed other genome-editing methods in a reporter system and at endogenous loci, efficiently creating large and precise genomic alterations. In a mouse model of tyrosinemia, PEDAR removed a 1.38-kb pathogenic insertion within the Fah gene and precisely repaired the deletion junction to restore FAH expression in liver.

Publication types

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

MeSH terms

  • Animals
  • CRISPR-Cas Systems* / genetics
  • Endonucleases / genetics
  • Gene Editing* / methods
  • Genome
  • Mice
  • RNA, Guide, Kinetoplastida / genetics


  • RNA, Guide
  • Endonucleases