Engineered Cpf1 variants with altered PAM specificities.

Nat Biotechnol
Authors
Keywords
Abstract

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.

Year of Publication
2017
Journal
Nat Biotechnol
Volume
35
Issue
8
Pages
789-792
Date Published
2017 Aug
ISSN
1546-1696
DOI
10.1038/nbt.3900
PubMed ID
28581492
PubMed Central ID
PMC5548640
Links
Grant list
DP1 MH100706 / MH / NIMH NIH HHS / United States
R01 MH110049 / MH / NIMH NIH HHS / United States
T32 GM007287 / GM / NIGMS NIH HHS / United States
T32 GM007753 / GM / NIGMS NIH HHS / United States