Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNAGln-processing system in human cells.
J Biol Chem
; 294(23): 9308-9315, 2019 06 07.
Article
em En
| MEDLINE
| ID: mdl-31010827
ABSTRACT
CRISPR/Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. High-fidelity Cas9 variants have been identified; however, they often have reduced activity, constraining their utility, which presents a major challenge for their use in research applications and therapeutics. Here we developed a tRNAGln-processing system to restore the activity of multiple high-fidelity Cas9 variants in human cells, including SpCas9-HF1, eSpCas9, and xCas9. Specifically, acting on previous observations that small guide RNAs (sgRNAs) harboring an extra A or G (A/G) in the first 5' nucleotide greatly affect the activity of high-fidelity Cas9 variants and that tRNA-sgRNA fusions improve Cas9 activity, we investigated whether a GN20 sgRNA fused to different tRNAs (G-tRNA-N20) could restore the activity of SpCas9 variants in human cells. Using flow cytometry, a T7E1 assay, deep sequencing-based DNA cleavage activity assays, and HEK-293 cells, we observed that a tRNAGln-sgRNA fusion system enhanced the activity of Cas9 variants, which could be harnessed for efficient correction of a pathogenic mutation in the retinoschisin 1 (RS1) gene, resulting in 6- to 8-fold improved Cas9 activity. We propose that the tRNA-processing system developed here specifically for human cells could facilitate high-fidelity Cas9-mediated human genome-editing applications.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA de Transferência de Glutamina
/
Sistemas CRISPR-Cas
/
Edição de Genes
Limite:
Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article