CRISPR-Cas9 zebrafish embryo editing
Materials Required
Principle
CRISPR-Cas9 zebrafish embryo editing introduces targeted double-strand breaks in genomic DNA by delivering Cas9 nuclease with a guide RNA into one-cell-stage embryos; repair by endogenous DNA-repair pathways produces indels or donor-mediated insertions that can be detected by phenotype, PCR-based genotyping, heteroduplex assays, Sanger sequencing, or amplicon sequencing[1][2][3][4]. The readout reflects the frequency and spectrum of edited alleles in mosaic F0 embryos or transmitted F1 animals; because injected embryos can carry multiple alleles, founder screening and sequence confirmation are required before establishing stable mutant lines[3][4][5][6].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Cas9 can be delivered as Cas9 mRNA or purified Cas9 protein; Cas9 protein with sgRNA forms an RNP that is active after injection, while Cas9 mRNA requires translation in the embryo[1][4][7][8].
• SgRNA is used to specify the genomic target, and guide choice is a major determinant of editing efficiency; CRISPRscan was developed from large-scale zebrafish sgRNA activity data to select efficient guides[5][6].
• PCR primers flanking the target site are used for genotyping edited embryos or founders by heteroduplex assay, fragment analysis, Sanger sequencing, or amplicon sequencing[6][9][10].
• Detection is typically DNA-based by PCR, heteroduplex mobility assay, fragment analysis, Sanger sequencing, or next-generation amplicon sequencing[6][9][10][11].
• A zebrafish microinjection setup is required to deliver Cas9 mRNA/sgRNA or Cas9 RNP into one-cell-stage embryos; published workflows use injection into the cell or yolk depending on the protocol[3][9][10].
• A thermal cycler, gel or fragment-analysis system, and sequencing access are used to detect indels and confirm edited alleles[6][9][10][11].
• SgRNA is used to specify the genomic target, and guide choice is a major determinant of editing efficiency; CRISPRscan was developed from large-scale zebrafish sgRNA activity data to select efficient guides[5][6].
• PCR primers flanking the target site are used for genotyping edited embryos or founders by heteroduplex assay, fragment analysis, Sanger sequencing, or amplicon sequencing[6][9][10].
• Detection is typically DNA-based by PCR, heteroduplex mobility assay, fragment analysis, Sanger sequencing, or next-generation amplicon sequencing[6][9][10][11].
• A zebrafish microinjection setup is required to deliver Cas9 mRNA/sgRNA or Cas9 RNP into one-cell-stage embryos; published workflows use injection into the cell or yolk depending on the protocol[3][9][10].
• A thermal cycler, gel or fragment-analysis system, and sequencing access are used to detect indels and confirm edited alleles[6][9][10][11].
Experimental Procedure
• Design sgRNAs against the target locus and prioritize guides with published zebrafish-specific scoring when possible; CRISPRscan was built from analysis of more than 1,000 zebrafish sgRNAs, and later work showed that unnecessary 5′ guanine additions can reduce activity in zebrafish embryos[5][6][12].
• Prepare Cas9 mRNA plus sgRNA or preassemble Cas9 protein with sgRNA as an RNP; both formats have been used successfully in zebrafish embryos, and RNP delivery has been reported to improve immediate activity and high-efficiency mutagenesis in some studies[1][4][7][8].
• Collect fertilized zebrafish embryos at the one-cell stage for injection, because early delivery reduces the number of unedited cell lineages and is the standard timing in zebrafish CRISPR embryo-editing studies[1][2][3][4].
• Inject the Cas9/sgRNA mixture into one-cell-stage embryos; reported zebrafish protocols include about 1 nL of 10 ng/µL sgRNA with 200 ng/µL Cas9 mRNA, about 2 nL of 100 ng/µL sgRNA mixture with 150 ng/µL Cas9 mRNA, and Cas9 protein/sgRNA RNP injection for higher direct nuclease delivery[4][7][9][10].
• Culture injected embryos and score survival, gross morphology, and any target-linked phenotype when such a phenotype is available; visible phenotypes were used in classic zebrafish CRISPR studies targeting pigmentation or reporter loci, but absence of phenotype does not exclude editing[1][3][4].
• Extract genomic DNA from injected embryos or fin clips of raised founders, amplify the target locus, and detect indels by heteroduplex mobility assay, fragment analysis, Sanger sequencing, or amplicon sequencing[6][9][10][11].
• Raise candidate F0 founders and outcross them to identify germline-transmitted alleles; direct germline screening and sequencing-based workflows were developed because F0 animals are mosaic[6][10][11].
• Use uninjected embryos as negative controls and, when feasible, a guide with a known visible or genotyping-validated target as a positive process control; editing efficiency should be estimated from embryo pools or individual embryos by PCR-based assays or sequencing rather than inferred only from survival or morphology[3][4][6][9][10].
• Interpret F0 phenotypes cautiously because injected embryos can be mosaic and can contain multiple alleles; stable mutant lines require identification of transmitted F1 alleles and sequence confirmation of the mutation[4][6][10][11].
• Prepare Cas9 mRNA plus sgRNA or preassemble Cas9 protein with sgRNA as an RNP; both formats have been used successfully in zebrafish embryos, and RNP delivery has been reported to improve immediate activity and high-efficiency mutagenesis in some studies[1][4][7][8].
• Collect fertilized zebrafish embryos at the one-cell stage for injection, because early delivery reduces the number of unedited cell lineages and is the standard timing in zebrafish CRISPR embryo-editing studies[1][2][3][4].
• Inject the Cas9/sgRNA mixture into one-cell-stage embryos; reported zebrafish protocols include about 1 nL of 10 ng/µL sgRNA with 200 ng/µL Cas9 mRNA, about 2 nL of 100 ng/µL sgRNA mixture with 150 ng/µL Cas9 mRNA, and Cas9 protein/sgRNA RNP injection for higher direct nuclease delivery[4][7][9][10].
• Culture injected embryos and score survival, gross morphology, and any target-linked phenotype when such a phenotype is available; visible phenotypes were used in classic zebrafish CRISPR studies targeting pigmentation or reporter loci, but absence of phenotype does not exclude editing[1][3][4].
• Extract genomic DNA from injected embryos or fin clips of raised founders, amplify the target locus, and detect indels by heteroduplex mobility assay, fragment analysis, Sanger sequencing, or amplicon sequencing[6][9][10][11].
• Raise candidate F0 founders and outcross them to identify germline-transmitted alleles; direct germline screening and sequencing-based workflows were developed because F0 animals are mosaic[6][10][11].
• Use uninjected embryos as negative controls and, when feasible, a guide with a known visible or genotyping-validated target as a positive process control; editing efficiency should be estimated from embryo pools or individual embryos by PCR-based assays or sequencing rather than inferred only from survival or morphology[3][4][6][9][10].
• Interpret F0 phenotypes cautiously because injected embryos can be mosaic and can contain multiple alleles; stable mutant lines require identification of transmitted F1 alleles and sequence confirmation of the mutation[4][6][10][11].
Troubleshooting
Problem: Low or absent editing.
• Possible cause: inefficient sgRNA.• Literature-supported solution: redesign and test additional sgRNAs using zebrafish-specific guide-efficiency information, because sgRNA activity varies strongly by target sequence and CRISPRscan was developed to improve zebrafish guide selection[4][5][6].
Problem: High embryo lethality after injection.
• Possible cause: excessive somatic disruption or toxic delivery conditions.• Literature-supported solution: reduce somatic Cas9 activity when stable-line generation is the goal, because germline-targeted Cas9 strategies were reported to increase viability by reducing somatic mutations[5][6].
Problem: F0 phenotype is difficult to interpret.
• Possible cause: mosaicism and multiple allele classes in injected embryos.• Literature-supported solution: confirm allele composition by sequencing and establish F1 carriers before drawing stable genotype-phenotype conclusions[4][6][10][11].
Problem: Genotyping misses complex or mixed alleles.
• Possible cause: heterogeneous indel populations in mosaic embryos or founders.• Literature-supported solution: use amplicon sequencing or sequencing-based founder screening when fragment or heteroduplex assays are insufficient[6][10][11].
References:
- [1]. Hwang WY, et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol. 2013;31(3):227-229. [Content Brief]
- [2]. Chang N, et al. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res. 2013;23(4):465-472. [Content Brief]
- [3]. Jao LE, et al. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci U S A. 2013;110(34):13904-13909. [Content Brief]
- [4]. Gagnon JA, et al. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS One. 2014;9(5):e98186. [Content Brief]
- [5]. Moreno-Mateos MA, et al. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods. 2015;12(10):982-988. [Content Brief]
- [6]. Vejnar CE, et al. Optimized CRISPR-Cas9 system for genome editing in zebrafish. Cold Spring Harb Protoc. 2016;2016(10):pdb.prot086850. [Content Brief]
- [7]. Burger A, et al. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes. Development. 2016;143(11):2025-2037. [Content Brief]
- [8]. Sung YH, et al. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res. 2014;24(1):125-131. [Content Brief]
- [9]. Varshney GK, et al. High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9. Genome Res. 2015;25(7):1030-1042. [Content Brief]
- [10]. Brocal I, et al. Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish. BMC Genomics. 2016;17:259. [Content Brief]
- [11]. Medishetti R, et al. CRISPR-Cas9-induced gene knockout in zebrafish. STAR Protoc. 2022;3(4):101779. [Content Brief]
- [12]. Hoshijima K, et al. Highly efficient CRISPR-Cas9-based methods for generating deletion mutations and F0 embryos that lack gene function in zebrafish. Dev Cell. 2019;51(5):645-657.e4. [Content Brief]
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