Methods for Measuring CRISPR/Cas9 DNA Cleavage in Cells.
Methods Mol Biol
; 2162: 197-213, 2021.
Article
en En
| MEDLINE
| ID: mdl-32926384
ABSTRACT
The CRISPR/Cas9 system has transformed how gene knockout and knock-in studies are performed in the lab, and it is poised to revolutionize medicine. However, one of the present limitations of this technology is its imperfect specificity. While CRISPR/Cas9 can be programmed to cut a specific DNA target sequence with relative precision, off-target sequence cleavage can occur in large genomes. Importantly, several techniques have recently been developed to measure CRISPR/Cas9 on- and off-target DNA cleavage in cells. Here, we present detailed protocols for evaluating the specificity of CRISPR/Cas9 and related systems in cells using both targeted-approaches, in which off-target sites are known a priori, and unbiased approaches which are able to identify off-target cleavage events throughout an entire genome. Together, these techniques can be used to assess the reliability of experimental models generated using CRISPR/Cas9 as well as the safety of therapeutics employing this technology.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Asociadas a CRISPR
/
Sistemas CRISPR-Cas
/
Edición Génica
/
Proteína 9 Asociada a CRISPR
Tipo de estudio:
Guideline
Límite:
Humans
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2021
Tipo del documento:
Article
País de afiliación:
Canadá
Pais de publicación:
EEUU
/
ESTADOS UNIDOS
/
ESTADOS UNIDOS DA AMERICA
/
EUA
/
UNITED STATES
/
UNITED STATES OF AMERICA
/
US
/
USA