Engineered prime editors with PAM flexibility

Mol Ther. 2021 Jun 2;29(6):2001-2007. doi: 10.1016/j.ymthe.2021.02.022. Epub 2021 Feb 24.


Although prime editors are a powerful tool for genome editing, which can generate various types of mutations such as nucleotide substitutions, insertions, and deletions in the genome without double-strand breaks or donor DNA, the conventional prime editors are still limited to their target scopes because of the PAM preference of the Streptococcus pyogenes Cas9 (spCas9) protein. Here, we describe the engineered prime editors to expand the range of their target sites using various PAM-flexible Cas9 variants. Using the engineered prime editors, we could successfully generate more than 50 types of mutations with up to 51.7% prime-editing activity in HEK293T cells. In addition, we successfully introduced the BRAF V600E mutation, which could not be induced by conventional prime editors. These variants of prime editors will broaden the applicability of CRISPR-based prime editing technologies in biological research.

Keywords: CRISPR-Cas; PAM variants; genome editing; prime editing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Amino Acid Substitution
  • Binding Sites
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Gene Editing*
  • Genetic Engineering* / methods
  • HEK293 Cells
  • Humans
  • Mutation
  • Nucleotide Motifs*
  • Proto-Oncogene Proteins B-raf / genetics


  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf
  • CRISPR-Associated Protein 9