Engineered Cpf1 variants with altered PAM specificities.
Nat Biotechnol
; 35(8): 789-792, 2017 Aug.
Article
em En
| MEDLINE
| ID: mdl-28581492
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 â¼11 bp.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
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Variação Genética
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Engenharia Genética
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Mutagênese Sítio-Dirigida
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Endonucleases
Limite:
Humans
Idioma:
En
Revista:
Nat Biotechnol
Ano de publicação:
2017
Tipo de documento:
Article