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SCIENCE CHINA Life Sciences, Volume 63, Issue 1: 59-67(2020) https://doi.org/10.1007/s11427-019-1607-9

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

Jia Li1,†,*, Hong-Yu Li1,2,†, Shan-Ye Gu3,†, Hua-Xing Zi1,2, Lai Jiang3, Jiu-Lin Du1,2,4,*
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  • ReceivedNov 2, 2019
  • AcceptedDec 16, 2019
  • PublishedDec 20, 2019

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 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 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.


Funded by

the Young Scientists Fund of the National Natural Science Foundation of China(31500849)

Shanghai Municipal Science and Technology Major Project(18JC1410100,2018SHZDZX05)

the Key Research Program of Frontier Sciences(QYZDY-SSW-SMC028)

the Strategic Priority Research Program(XDB32010200)

the International Partnership Program

Bureau of International Co-operation of Chinese Academy of Sciences(153D31KYSB20170059)

China Wan-Ren Program

and Shanghai Leading Scientist Program.


Acknowledgment

We are grateful to Drs. N. Lawson for providing the Tg(flk1:EGFP) line, D. Traver for providing the Tg(bactin2:loxP-STOP-loxP-DsRedEx) line and K. Kikuchi for providing the pZwitch+1 plasmid. This work was supported by the Young Scientists Fund of the National Natural Science Foundation of China (31500849), Shanghai Municipal Science and Technology Major Project (18JC1410100, 2018SHZDZX05), the Key Research Program of Frontier Sciences (QYZDY-SSW-SMC028), the Strategic Priority Research Program (XDB32010200) of Chinese Academy of Sciences, the International Partnership Program, Bureau of International Co-operation of Chinese Academy of Sciences (153D31KYSB20170059), China Wan-Ren Program, and Shanghai Leading Scientist Program.


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

Figure S1 Sequencing analysis of the genome and transcript of hey2zCKOIS.

Figure S2 Genotyping and expression of hey2zCKOIS.

Figure S3 Genotyping of hey2zCKOIS;Ki(flk1-P2A-Cre) and projected confocal images of hey2zCKOIS/zCKOIS and Ki(flk1-P2A-Cre).

Table S1 Sequences of the primers for constructing the hey2zCKOIS donor plasmid

Table S2 Sequences of the primers for genotyping of the hey2zCKOIS and hey2zCKOIS-inv genomic loci and the transcripts in Figure 1B and C, Figure 2C and D, and Figure S3A in Supporting Information

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    Generation of a hey2zCKOIS zebrafish line. A, Schematic of the intron targeting-mediated strategy for generating hey2zCKOIS zebrafish by using the CRISPR/Cas9 system. The zebrafish hey2 has 5 exons, and E4 and E5 represent the 4th and 5th exons, respectively. The hey2zCKOIS donor was integrated into the hey2 locus after co-injection of the donor with the sgRNA and zCas9 mRNA. The sgRNA target sequence is shown in red and the protospacer adjacent motif (PAM) sequence in green. The left and right arm sequences of the donor plasmids are indicated by the brown lines with double arrows. The left arm in the donor is 3,300 bp in length, including the original left arm in the genome and an inverted TagRFP cassette sequence. The right arm is 1,107 bp in length. GSG-P2A is glycine-serine-glycine-P2A sequence. B, PCR analysis of the genomic DNA obtained from the F1 progenies of the hey2zCKOIS founder. A 3.2 kb band was amplified by using the F1 and R1 primers, and a 5.3 kb band was amplified by using the F1 and R2 primers. The two bands are only present in hey2zCKOIS embryos, but not in the wild-type (WT) group. The F1, R1, F2, and R2 primers are indicated in (A). C, Left, RT-PCR analysis using the cDNA of hey2zCKOIS F1 embryos. A 1.5 kb band amplified by the F1 and R1 primers only appears in hey2zCKOIS embryos. Right, control RT-PCR using the primers binding hey2 coding sequence showed a 0.3 kb band. D, Projected confocal images (lateral view) of a hey2zCKOIS;Ki(GFAP-TagBFP) embryos at 1.5 dpf, showing EGFP expression in glial cells in the brain labeled by TagBFP. Ki(GFAP-TagBFP) is a KI line with TagBFP expression specific in glial cells. White arrowheads, the midbrain; arrow, the forebrain. Scale bar, 100 μm. E, Projected confocal images (lateral view) of a Ki(GFAP-TagBFP);hey2zCKOIS embryo at 3.5 dpf, showing EGFP expression in the glial cells at the spinal cord (white arrow heads). Cyan arrowheads, non-specific signals on the skin. Scale bar, 100 μm. F and G, Projected confocal imaging of the hey2zCKOIS;Tg(flk1:Ras-mCherry) embryos at 3.5 dpf. F, EGFP expression in the basal communicating artery (BCA) and postcardinal communicating segment (PCS) in the brain (dorsal view). Top left, EGFP signal; top right, merged signals (EGFP/mCherry). The outlined areas labeled a and b in the top right panel are enlarged below. White arrowheads, BCA and PCS; white arrows, choroidal vascular plexus (CVP). G, EGFP expression in the dorsal aorta (DA) but not in the postcardinal vein (PCV) in the trunk (lateral view). Arterial intersegmental vessels (aISVs) extending from the DA exhibited more EGFP signal than venous ISVs (vISVs) extending from the PCV. Top left, EGFP signal; top right, merged signals (EGFP/mCherry). The outlined areas labeled a and b in the top right panel are enlarged below. White arrowheads, DA and aISVs; white arrows, PCV and vISVs. Cyan arrowheads, non-specific signals on the skin. Scale bar, 100 μm.

  • Figure 2

    Characterization of the hey2zCKOIS-inv Allele. A, Schematic of the hey2zCKOIS allele and the translated Hey2zCKOIS protein. The endogenous Hey2 protein includes an N-terminal bHLH domain, an Orange domain, and a protein-protein interaction YRPW (“Y”) motif near the C-terminus. The Hey2zCKOIS protein is a fusion of the wild-type (WT) Hey2 protein and a GSG-P2A-EGFP. B, Schematic of the hey2zCKOIS-inv allele produced via Cre-induced inversion of the hey2zCKOIS allele. The translated Hey2zCKOIS-inv protein is a truncated Hey2 with only the bHLH domain fused to a P2A-TagRFP. C, PCR detection of the inversion in hey2zCKOIS-inv genome. A 2.8 kb band was only present in the hey2zCKOIS-inv but not in the WT or hey2zCKOIS embryos. D, Left, RT-PCR analysis for detection of the transcription of hey2zCKOIS-inv. A 0.9 kb band was amplified only in the hey2zCKOIS-inv, not in the WT or hey2zCKOIS embryos. Right, control RT-PCR using primers binding hey2 coding sequence showed a 0.3 kb band in all groups. E, Projected confocal images (lateral view) of a hey2zCKOIS-inv;Ki(GFAP-TagBFP) embryo at 1.5 dpf, showing TagRFP expression in glial cells. The boxed area is enlarged on the right. White arrow heads, the midbrain; arrow, the forebrain. Scale bar, 100 μm. F, Projected confocal images (lateral view) of the trunk of a hey2zCKOIS/zCKOIS-inv embryo at 2.5 dpf. The red fluorescence is encoded by the truncated Hey2-P2A-TagRFP in the hey2zCKOIS-inv allele and the green fluorescence is encoded by the WT Hey2-GSG-P2A-EGFP in the hey2zCKOIS allele. Cyan arrowheads, non-specific signals on the skin. Scale bar, 100 μm. G, Confocal images of the DA in a hey2zCKOIS/zCKOIS-inv embryo at 2.5 dpf showing two HSCs budding from the DA. Scale bar, 50 μm. H, Bright-field images showing severe pericardial edema in the homozygous hey2zCKOIS-inv/zCKOIS-in (top) but not in the heterozygous hey2zCKOIS-inv (bottom) at 3.5 dpf. Left, merged images of bright field (BF) and TagRFP. Right, TagRFP. Scale bar, 500 μm. I, Percentage of embryos with different genetic backgrounds showing normal circulation to the tail. The number on the bar is the total number of 3.5 dpf embryos examined.

  • Figure 3

    EC-specific KO of hey2. A, Schematic of the strategy for endothelial cell (EC)-specific KO of hey2. The Hey2 protein in ECs will be destroyed in the embryo carrying homozygous hey2zCKOIS alleles and Ki(flk1-P2A-Cre) background. In non-ECs where Cre is not expressed, the hey2zCKOIS will not be inverted and thus the WT Hey2 protein is expressed. B, RT-PCR analysis using the cDNA for detection of the transcripts of hey2zCKOIS and hey2zCKOIS-inv. Left, a 1.5 kb band was amplified by the F1 and R2 primers in both hey2zCKOIS;Ki(flk1-P2A-Cre) and hey2zCKOIS but not in the WT embryos. Middle, a 0.9 kb band amplified by the F1 and F2 primers was only present in the hey2zCKOIS;Ki(flk1-P2A-Cre) embryos. Right, control RT-PCR using primers binding hey2 coding sequence showed a 0.3 kb band in each group. C, Projected confocal images (lateral view) of trunk vessels in a hey2zCKOIS/zCKOIS;Ki(flk1-P2A-Cre) embryos at 3.5 dpf. Cre-induced inversion of the hey2zCKOIS enables the TagRFP expression in the DA and aISVs, where EGFP expression is barely deleted (arrowheads and dashed lines). Top left, EGFP signal; top right, merged signals (EGFP/TagRFP). The outlined area is enlarged below. Cyan arrowheads, non-specific signals on the skin. Scale bar, 100 μm. D, Percentage of the embryos with different genetic backgrounds showing normal circulation to the tail at 3.5 dpf. The number on the bar is the total number of fish examined.

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