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. 2022 Oct 26;3(4):101779.
doi: 10.1016/j.xpro.2022.101779. eCollection 2022 Dec 16.

CRISPR-Cas9-induced gene knockout in zebrafish

Raghavender Medishetti  1 , Keerthana Balamurugan  2 , Krishnaveni Yadavalli  3 , Rita Rani  3 , Aarti Sevilimedu  3 , Anil Kumar Challa  3 , Kishore Parsa  3 , Kiranam Chatti  4
Affiliations

CRISPR-Cas9-induced gene knockout in zebrafish

Raghavender Medishetti et al. STAR Protoc. .

Abstract

The application of CRISPR has greatly facilitated genotype-phenotype studies of human disease models. In this protocol, we describe CRISPR-Cas9-induced gene knockout in zebrafish, utilizing purified Cas9 protein and in vitro-transcribed sgRNA. This protocol targets the PHLPP1 gene in an Indian wild-caught strain, but is broadly applicable. Major factors influencing protocol success include zebrafish health and fecundity, sgRNA efficiency and specificity, germline transmission, and mutant viability. For complete details on the use and execution of this protocol, please refer to Balamurugan et al. (2022).

Keywords: CRISPR; Genetics; Model organisms; Molecular biology; Sequencing.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Screenshot of the Benchling web application used to design sgRNAs
Figure 2
Figure 2
sgRNA synthesis template and schematic Top Panel: Schematic of the oligonucleotides used to generate the template for sgRNA synthesis; Bottom Panel: Schematic of the annealed and extended sgRNA template, ready to be used for sgRNA synthesis via T7-driven in vitro transcription.
Figure 3
Figure 3
FemtoJet microinjection system (Eppendorf) with the microinjection settings shown
Figure 4
Figure 4
Zebrafish embryo microinjection Top Panel: View of zebrafish embryos during microinjection. The Phenol Red dye used as an additive for visualization is seen; Bottom Panel: Close-up view of zebrafish embryos during microinjection.
Figure 5
Figure 5
Cas9 protein purification Left Panel: Ni-Affinity chromatography of Cas9 protein. 1: Ladder. 2: Flow-through. 3: Wash. 4–9: Elutions. 10: Beads; Right Panel: Cation exchange chromatography of Cas9 protein following Ni-Affinity chromatography. 1: Ladder. 2: Flow-through. 3–10: Elutions.
Figure 6
Figure 6
In vitro CRISPR catalytic activity assessment (Cas9-sgRNA RNP complex) 1: Control. 2: sgRNA 1. 3: sgRNA 2. 4: sgRNA 1+2. 5: Control. 6: Ladder.
Figure 7
Figure 7
HMA genotyping of F0 and F1 embryos Left Panel: HMA of F0 embryos (CRISPR-injected embryos).1: Embryo 1 (HMA-Positive). 2: Ladder. 3: Embryo 2 (HMA-Positive). 4: Embryo 3 (HMA-Positive). 5: Embryo 4. 6: Embryo 5. 7: Embryo 6. 8: Embryo 7. 9: Embryo 8. 10: Control; Right Panel: HMA of F1 embryos (progeny of F0 adults and WT). 1: Control. 2: Embryo 1 (HMA-Positive). 3: Embryo 2 (HMA-Positive). 4: Embryo 3 (HMA-Positive). 5: Embryo 4. 6: Embryo 5 (HMA-Positive). 7: Embryo 6. 8: Embryo 7 (HMA-Positive). 9: Embryo 8 (HMA-Positive). 10: Ladder.
Figure 8
Figure 8
HMA genotyping of F2 embryos Left Panel: HMA of F2 embryos (progeny of F1 adults) without denaturation and annealing. 1: Ladder. 2: Embryo 1 (WT Control). 3: Embryo 2. 4: Embryo 3. 5: Embryo 4. 6: Embryo 5. 7: Embryo 6. 8: Embryo 7. 9: Embryo 8; Right Panel: HMA of F2 embryos with denaturation and annealing. 1: Ladder. 2: Embryo 1 (WT Control). 3: Embryo 2. 4: Embryo 3 (HMA-Positive). 5: Embryo 4 (HMA-Positive). 6: Embryo 5. 7: Embryo 6. 8: Embryo 7. 9: Embryo 8 (HMA-Positive). The F2 HMA above shows that embryos 3, 4, and 8 are homozygous mutants. WT F2 samples (embryos 2, 5, 6, and 7) yield only a single band under both the above conditions. All the HMA data shown above is with embryos. Adult samples (tail or fin clips) is expected to yield similar results.
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References

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