One-step Generation of Zebrafish Carrying a Conditional Knockout-Knockin Visible Switch via CRISPR/Cas9-Mediated Intron Targeting

SUMMARYThe zebrafish has been becoming a popular vertebrate animal model in biomedical research. However, it is still challenging for making conditional gene knockout (CKO) 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 hey2zCKOIS which served as a hey2 KI reporter with EGFP expression. Upon Cre induction in targeted cells, the hey2zCKOIS was switched to a non-functional CKO allele hey2zCKOIS-inv associated with TagRFP expression, enabling to visualize CKO alleles. Thus, the simplification of the design, and the visibility and combination of both CKO and KI alleles’ engineering make our zCKOIS strategy an applicable CKO approach for zebrafish.

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One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs

Cell Research ◽

10.1038/cr.2017.81 ◽

2017 ◽

Vol 27 (7) ◽

pp. 933-945 ◽

Cited By ~ 77

Author(s):

Erwei Zuo ◽

Yi-Jun Cai ◽

Kui Li ◽

Yu Wei ◽

Bang-An Wang ◽

...

Keyword(s):

Knockout Mice ◽

Gene Editing ◽

Gene Knockout ◽

Gene Knockout Mice ◽

One Step ◽

Step Generation

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One-step generation of myostatin gene knockout sheep via the CRISPR/Cas9 system

Frontiers of Agricultural Science and Engineering ◽

10.15302/j-fase-2014007 ◽

2014 ◽

Vol 1 (1) ◽

pp. 2 ◽

Cited By ~ 35

Author(s):

HAN Hongbing ◽

MA Yonghe ◽

WANG Tao ◽

LIAN Ling ◽

TIAN Xiuzhi ◽

...

Keyword(s):

Gene Knockout ◽

Myostatin Gene ◽

One Step ◽

Step Generation

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One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish

eLife ◽

10.7554/elife.48081 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 2

Author(s):

Wenyuan Li ◽

Yage Zhang ◽

Bingzhou Han ◽

Lianyan Li ◽

Muhang Li ◽

...

Keyword(s):

Conditional Knockout ◽

Dual Function ◽

Fluorescent Reporter ◽

Knock Out ◽

Efficient Generation ◽

Before And After ◽

One Step ◽

Low Efficiency ◽

Genetic Compensation ◽

Genome Modifications

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.

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