生物
基因组工程
DNA
核酸内切酶
突变
遗传学
人类基因组
计算生物学
分子生物学
基因组
基因组编辑
化学
突变体
基因
作者
Linyi Gao,David Benjamin Turitz Cox,Wang‐Ji Yan,John C. Manteiga,Martin Schneider,Takashi Yamano,Hiroshi Nishimasu,Osamu Nureki,Nicola Crosetto,Feng Zhang
摘要
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 cells1,2,3,4,5,6,7. 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 BLISS7 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.
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