Engineered Cpf1 variants with altered PAM specificities

Nat Biotechnol. 2017 Aug;35(8):789-792. doi: 10.1038/nbt.3900. Epub 2017 Jun 5.


The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.

MeSH terms

  • Acidaminococcus / enzymology
  • Acidaminococcus / genetics
  • Bacterial Proteins / genetics*
  • Endonucleases / genetics*
  • Genetic Engineering / methods*
  • Genetic Variation / genetics*
  • HEK293 Cells
  • Humans
  • Mutagenesis, Site-Directed / methods*


  • Bacterial Proteins
  • Endonucleases