Highly Parallel Profiling of Cas9 Variant Specificity.
Mol Cell
; 78(4): 794-800.e8, 2020 05 21.
Article
en En
| MEDLINE
| ID: mdl-32187529
ABSTRACT
Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Variación Genética
/
ARN Guía de Kinetoplastida
/
Roturas del ADN de Doble Cadena
/
Sistemas CRISPR-Cas
/
Edición Génica
/
Proteína 9 Asociada a CRISPR
Límite:
Humans
Idioma:
En
Revista:
Mol Cell
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2020
Tipo del documento:
Article