One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish
- Peking University, China
- Peking University Shenzhen Graduate School, China
- Gcrispr (Tianjin) Genetic Technology, China
Abstract
CRISPR/Cas systems are widely used to knockout 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.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files.
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Funding
National Key Research and Development Program of China (2018YFA0801000)
- Bo Zhang
National Key Research and Development Program of China (2016YFA0100500)
- Bo Zhang
National Key Basic Research Program of China (2015CB942803)
- Bo Zhang
National Natural Science Foundation of China (31671500)
- Bo Zhang
National Natural Science Foundation of China (31871458)
- Bo Zhang
National Natural Science Foundation of China (81371264)
- Bo Zhang
PKU Qidong-SLS Innovation Fund
- Bo Zhang
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal experiments were approved by Institutional Animal Care and Use Committee (IACUC) of Peking University. The reference from IACUC of Peking University is LSC-ZhangB-2.
Copyright
© 2019, Li et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish
eLife 8:e48081.
https://doi.org/10.7554/eLife.48081
