A Tandem Guide RNA-Based Strategy for Efficient CRISPR Gene Editing of Cell Populations with Low Heterogeneity of Edited Alleles.
CRISPR J
; 3(2): 123-134, 2020 04.
Article
em En
| MEDLINE
| ID: mdl-32315231
ABSTRACT
CRISPR/Cas9-based gene knockouts (KOs) enable precise perturbation of target gene function in human cells, which is ideally assessed in an unbiased fashion by molecular omics readouts. Typically, this requires the lengthy process of isolating KO subclones. We show here that KO subclones are phenotypically heterogenous, regardless of the guide RNA used. We present an experimental strategy that avoids subcloning and achieves fast and efficient gene silencing on cell pools, based on the synergistic combination of two guide RNAs mapping at close (40-300 bp) genomic proximity. Our strategy results in better predictable indel generation with a low allelic heterogeneity, concomitant with low or undetectable residual target protein expression, as determined by MS3 mass spectrometry proteomics. Our method is compatible with nondividing primary cells and can also be used to study essential genes. It enables the generation of high confidence omics data which solely reflect the phenotype of the target ablation.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Técnicas de Inativação de Genes
/
Sistemas CRISPR-Cas
/
Edição de Genes
Limite:
Animals
/
Humans
Idioma:
En
Revista:
CRISPR J
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Alemanha