One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish.
Elife
; 82019 10 30.
Article
en En
| MEDLINE
| ID: mdl-31663848
ABSTRACT
CRISPR/Cas systems are widely used to knock out genes by inducing indel mutations, which are prone to genetic compensation. Complex genome modifications such as knockin (KI) might bypass compensation, though difficult to practice due to low efficiency. Moreover, no 'two-in-one' KI strategy combining conditional knockout (CKO) with fluorescent gene-labeling or further allele-labeling has been reported. Here, we developed a dual-cassette-donor strategy and achieved one-step and efficient generation of dual-function KI alleles at tbx5a and kctd10 loci in zebrafish via targeted insertion. These alleles display fluorescent gene-tagging and CKO effects before and after Cre induction, respectively. By introducing a second fluorescent reporter, geno-tagging effects were achieved at tbx5a and sox10 loci, exhibiting CKO coupled with fluorescent reporter switch upon Cre induction, enabling tracing of three distinct genotypes. We found that LiCl purification of gRNA is critical for highly efficient KI, and preselection of founders allows the efficient germline recovery of KI events.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Coloración y Etiquetado
/
Pez Cebra
/
Alelos
/
Técnicas de Inactivación de Genes
/
Edición Génica
Límite:
Animals
Idioma:
En
Revista:
Elife
Año:
2019
Tipo del documento:
Article
País de afiliación:
China