One-step generation of zebrafish carrying a conditional knockout-knockin visible switch via CRISPR/Cas9-mediated intron targeting

Jia Li; Hong-Yu Li; Shan-Ye Gu; Hua-Xing Zi; Lai Jiang; Jiu-Lin Du

doi:10.1007/s11427-019-1607-9

One-step generation of zebrafish carrying a conditional knockout-knockin visible switch via CRISPR/Cas9-mediated intron targeting

Sci China Life Sci. 2020 Jan;63(1):59-67. doi: 10.1007/s11427-019-1607-9. Epub 2019 Dec 20.

Authors

Jia Li^#¹, Hong-Yu Li^#^{2

3}, Shan-Ye Gu^#⁴, Hua-Xing Zi^{2

3}, Lai Jiang⁴, Jiu-Lin Du^{5

6

7}

Affiliations

¹ Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Chinese Academy of Sciences, Shanghai, 200031, China. lijia@ion.ac.cn.
² Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Chinese Academy of Sciences, Shanghai, 200031, China.
³ University of Chinese Academy of Sciences, Beijing, 100049, China.
⁴ Department of Anesthesiology and Surgical Intensive Care Unit, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, China.
⁵ Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Chinese Academy of Sciences, Shanghai, 200031, China. forestdu@ion.ac.cn.
⁶ University of Chinese Academy of Sciences, Beijing, 100049, China. forestdu@ion.ac.cn.
⁷ School of Life Science and Technology, ShanghaiTech University, Shanghai, 200031, China. forestdu@ion.ac.cn.

^# Contributed equally.

PMID: 31872378
DOI: 10.1007/s11427-019-1607-9

Abstract

The zebrafish has become a popular vertebrate animal model in biomedical research. However, it is still challenging to make conditional gene knockout (CKO) models in zebrafish due to the low efficiency of homologous recombination (HR). Here we report an efficient non-HR-based method for generating zebrafish carrying a CKO and knockin (KI) switch (zCKOIS) coupled with dual-color fluorescent reporters. Using this strategy, we generated hey2^zKOIS which served as a hey2 KI reporter with EGFP expression. Upon Cre induction in targeted cells, the hey2^zCKOIS was switched to a non-functional CKO allele hey2^zCKOIS-invassociated with TagRFP expression, enabling visualization of the CKO alleles. Thus, simplification of the design, and the visibility and combination of both CKO and KI alleles make our zCKOIS strategy an applicable CKO approach for zebrafish.

Keywords: NHEJ; conditional knockout; knockin; non-HR; visible switch; zCKOIS; zebrafish.

MeSH terms

Alleles
Animals
Animals, Genetically Modified
Base Sequence
Basic Helix-Loop-Helix Transcription Factors / genetics*
CRISPR-Cas Systems / genetics*
Gene Expression
Gene Knock-In Techniques / methods
Gene Knockout Techniques / methods
Gene Targeting / methods*
Genetic Engineering
Genotype
Green Fluorescent Proteins / genetics
Homologous Recombination / genetics*
Introns / genetics*
Zebrafish
Zebrafish Proteins / genetics*

Substances

Basic Helix-Loop-Helix Transcription Factors
Zebrafish Proteins
hey2 protein, zebrafish
Green Fluorescent Proteins