High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects.
Nat Biotechnol
; 41(1): 108-119, 2023 01.
Article
em En
| MEDLINE
| ID: mdl-35953673
ABSTRACT
CRISPR-Cas13 systems have recently been used for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has limited their in vivo applications. Here, we design a dual-fluorescence reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants including Cas13d and Cas13X exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. High-fidelity Cas13 variants show similar RNA knockdown activity to wild-type Cas13 but no detectable collateral damage in transgenic mice or adeno-associated-virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for targeted degradation of RNAs in basic research and therapeutic applications.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
RNA
/
Sistemas CRISPR-Cas
Limite:
Animals
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article