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Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio
Published on: August 28, 2018
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One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish
Wenyuan Li1, Yage Zhang1, Bingzhou Han1
1Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, Peking University Genome Editing Research Center, College of Life Sciences, Peking University, Beijing, China.
Elife
|October 31, 2019
Summary
This study introduces an efficient CRISPR/Cas9 dual-cassette knockin strategy in zebrafish. This method enables simultaneous gene tagging and conditional knockout, overcoming limitations of traditional gene editing.
Area of Science:
- Molecular Biology
- Genetics
- Zebrafish Model Organisms
Background:
- CRISPR/Cas systems are effective for gene knockout via indels but can trigger genetic compensation.
- Complex genomic modifications like knockin (KI) are challenging due to low efficiency and lack of combined functionalities.
- Existing methods do not offer a 'two-in-one' strategy for simultaneous conditional knockout (CKO) and fluorescent gene-labeling.
Purpose of the Study:
- To develop an efficient, one-step dual-cassette knockin strategy in zebrafish.
- To create dual-function knockin alleles combining conditional knockout with fluorescent gene-tagging.
- To enable tracing of multiple genotypes through inducible fluorescent reporter switches.
Main Methods:
- Developed a dual-cassette-donor strategy for targeted gene insertion in zebrafish.
- Applied the strategy to generate knockin alleles at tbx5a and kctd10 loci.
- Utilized LiCl purification of gRNA and founder preselection for enhanced efficiency and germline recovery.
Main Results:
- Achieved efficient, one-step generation of dual-function knockin alleles at tbx5a and kctd10.
- Demonstrated fluorescent gene-tagging and conditional knockout (CKO) effects upon Cre induction.
- Successfully created alleles with CKO and inducible fluorescent reporter switches for tracing three distinct genotypes at tbx5a and sox10 loci.
Conclusions:
- The dual-cassette-donor strategy enables efficient generation of complex, dual-function knockin alleles.
- This approach overcomes limitations of traditional gene knockout and provides advanced genetic manipulation tools.
- LiCl purification of gRNA and founder preselection are critical for maximizing knockin efficiency and germline transmission.
