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Engineered Cpf1 variants with altered PAM specificities
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Journal Article
Engineered Cpf1 variants with altered PAM specificities
2017
Overview
The targeting range of the CRISPR endonuclease Cpf1 is increased three-fold by molecular engineering.
The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells
1
,
2
,
3
,
4
,
5
,
6
,
7
. 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
7
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.
Publisher
Nature Publishing Group US,Nature Publishing Group
Subject
