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One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish
- Source :
- eLife, Vol 8 (2019), eLife
- Publication Year :
- 2019
- Publisher :
- eLife Sciences Publications Ltd, 2019.
-
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.
- Subjects :
- 0301 basic medicine
Genotype
QH301-705.5
Science
Computational biology
Biology
Genome
General Biochemistry, Genetics and Molecular Biology
Germline
conditional knock out
03 medical and health sciences
Gene Knockout Techniques
0302 clinical medicine
Conditional gene knockout
CRISPR
Animals
Allele
Biology (General)
Indel
knockin
Gene
Zebrafish
Alleles
NHEJ
Gene Editing
General Immunology and Microbiology
Staining and Labeling
General Neuroscience
Genetics and Genomics
General Medicine
Zebrafish Proteins
biology.organism_classification
Tools and Resources
030104 developmental biology
CRISPR/cas
targeted insertion
gene-labeling
Medicine
030217 neurology & neurosurgery
Transcription Factors
Subjects
Details
- Language :
- English
- Volume :
- 8
- Database :
- OpenAIRE
- Journal :
- eLife
- Accession number :
- edsair.doi.dedup.....8cffa1f87080e0d7b5c894761620a30b