Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements.
Genome Biol
; 16: 253, 2015 Nov 19.
Article
em En
| MEDLINE
| ID: mdl-26585795
ABSTRACT
To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid libraries containing a randomized PAM as a function of Cas9-guide RNA complex concentration. Using this method, we accurately reproduce the canonical PAM preferences for Streptococcus pyogenes, Streptococcus thermophilus CRISPR3 (Sth3), and CRISPR1 (Sth1). Additionally, PAM and sgRNA solutions for a novel Cas9 protein from Brevibacillus laterosporus are provided by the assay and are demonstrated to support functional activity in vitro and in plants.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Marcação de Genes
/
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
/
Sistemas CRISPR-Cas
Tipo de estudo:
Clinical_trials
Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article