Enhancement of target specificity of CRISPR-Cas12a by using a chimeric DNA-RNA guide.
Nucleic Acids Res
; 48(15): 8601-8616, 2020 09 04.
Article
en En
| MEDLINE
| ID: mdl-32687187
ABSTRACT
The CRISPR-Cas9 system is widely used for target-specific genome engineering. CRISPR-Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Bacterianas
/
ADN
/
ARN Guía de Kinetoplastida
/
Endodesoxirribonucleasas
/
Proteínas Asociadas a CRISPR
/
Sistemas CRISPR-Cas
Límite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2020
Tipo del documento:
Article