DNA targeting specificity of RNA-guided Cas9 nucleases.
Nat Biotechnol
; 31(9): 827-32, 2013 Sep.
Article
em En
| MEDLINE
| ID: mdl-23873081
ABSTRACT
The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fatores de Transcrição
/
DNA
/
Engenharia Genética
/
Desoxirribonucleases
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Nat Biotechnol
Ano de publicação:
2013
Tipo de documento:
Article