CRISPR-Cas9 mouse zygote editing
Materials Required
Principle
CRISPR-Cas9 mouse zygote editing introduces Cas9 nuclease and guide RNA into one-cell embryos so that Cas9 creates a guide-directed double-strand break at the target locus; repair by non-homologous end joining can generate indels, while repair with an added donor template can generate defined knock-in or point-mutation alleles[1][2][3]. The readout is embryo, pup, or founder genotype, usually assessed by PCR, restriction-fragment analysis, Sanger sequencing, TIDE/sequence-trace analysis, or targeted sequencing; successful editing is interpreted as the presence of indels, intended HDR alleles, or both at the target locus[3][4][5].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use Cas9 mRNA plus sgRNA, Cas9 protein plus sgRNA, or Cas9 protein assembled with synthetic crRNA/tracrRNA; published mouse zygote studies support both microinjection and electroporation delivery formats[1][3][4][5][6][7].
• Use ssODN donor templates for point mutations or small insertions, and plasmid or larger donor templates only when the cited study specifically supports that design[3][5][8][9].
• Genotyping reagents include PCR primers flanking the target locus, restriction enzymes when an engineered restriction site is used, and Sanger or targeted-sequencing reagents for allele identification[3][5][6].
• Supported equipment includes micromanipulation and microinjection systems for pronuclear or cytoplasmic delivery, electroporators for zygote electroporation, embryo culture equipment, embryo-transfer equipment, and standard PCR/sequencing instruments for genotyping[2][3][4][5][6][10].
• Use ssODN donor templates for point mutations or small insertions, and plasmid or larger donor templates only when the cited study specifically supports that design[3][5][8][9].
• Genotyping reagents include PCR primers flanking the target locus, restriction enzymes when an engineered restriction site is used, and Sanger or targeted-sequencing reagents for allele identification[3][5][6].
• Supported equipment includes micromanipulation and microinjection systems for pronuclear or cytoplasmic delivery, electroporators for zygote electroporation, embryo culture equipment, embryo-transfer equipment, and standard PCR/sequencing instruments for genotyping[2][3][4][5][6][10].
Experimental Procedure
• Design guide RNAs against the genomic target and select a donor strategy according to the intended edit: NHEJ-based knockout, ssODN-mediated point mutation or small insertion, or cassette knock-in when supported by the selected delivery method[1][3][5][8][9].
• Prepare fertilized one-cell embryos, assemble the CRISPR reagent format selected for delivery, and use intact zygotes for electroporation protocols where intact-zona delivery was demonstrated[3][4][5][6][7].
• For microinjection, deliver CRISPR components into the pronucleus or cytoplasm of one-cell embryos; comparative work reported that RNA cytoplasmic injection gave better overall knockout output than plasmid pronuclear injection among tested microinjection modes[2].
• For electroporation, place zygotes in an electroporation-compatible medium containing CRISPR components and apply the published pulse program appropriate to the selected method; reported mouse zygote electroporation approaches include Cas9 mRNA/sgRNA delivery, Cas9 protein/sgRNA RNP delivery, serial electroporation, and synthetic dual-guide RNA/Cas9 RNP delivery[3][4][5][6][7].
• Where using the EEZy approach in C57BL/6 zygotes, the reported condition was two 3-ms pulses at 30 V with synthetic CRISPR/Cas9 components, and the study reported that acidic Tyrode’s zona weakening was dispensable for efficient editing under that RNP-based workflow[6].
• After delivery, culture embryos to the appropriate preimplantation stage for in vitro genotyping or transfer viable embryos into pseudopregnant recipients to generate founders, as reported in mouse zygote editing workflows[1][2][3][4][5].
• Analyze blastocysts, pups, or founder mice by PCR-based genotyping followed by restriction analysis when applicable, Sanger sequencing, TIDE analysis, or targeted sequencing to distinguish wild-type, indel, mosaic, and intended HDR alleles[3][5][6][7].
• Use untreated or wild-type embryos/mice as negative controls and include no-template PCR controls during genotyping; when a donor introduces a diagnostic restriction site, restriction-fragment analysis can serve as a locus-specific screening readout before sequence confirmation[5][6].
• Assess founder mosaicism and germline transmission by breeding selected founders and genotyping F1 offspring, because somatic genotyping alone may not fully establish germline genotype[5][7].
• Prepare fertilized one-cell embryos, assemble the CRISPR reagent format selected for delivery, and use intact zygotes for electroporation protocols where intact-zona delivery was demonstrated[3][4][5][6][7].
• For microinjection, deliver CRISPR components into the pronucleus or cytoplasm of one-cell embryos; comparative work reported that RNA cytoplasmic injection gave better overall knockout output than plasmid pronuclear injection among tested microinjection modes[2].
• For electroporation, place zygotes in an electroporation-compatible medium containing CRISPR components and apply the published pulse program appropriate to the selected method; reported mouse zygote electroporation approaches include Cas9 mRNA/sgRNA delivery, Cas9 protein/sgRNA RNP delivery, serial electroporation, and synthetic dual-guide RNA/Cas9 RNP delivery[3][4][5][6][7].
• Where using the EEZy approach in C57BL/6 zygotes, the reported condition was two 3-ms pulses at 30 V with synthetic CRISPR/Cas9 components, and the study reported that acidic Tyrode’s zona weakening was dispensable for efficient editing under that RNP-based workflow[6].
• After delivery, culture embryos to the appropriate preimplantation stage for in vitro genotyping or transfer viable embryos into pseudopregnant recipients to generate founders, as reported in mouse zygote editing workflows[1][2][3][4][5].
• Analyze blastocysts, pups, or founder mice by PCR-based genotyping followed by restriction analysis when applicable, Sanger sequencing, TIDE analysis, or targeted sequencing to distinguish wild-type, indel, mosaic, and intended HDR alleles[3][5][6][7].
• Use untreated or wild-type embryos/mice as negative controls and include no-template PCR controls during genotyping; when a donor introduces a diagnostic restriction site, restriction-fragment analysis can serve as a locus-specific screening readout before sequence confirmation[5][6].
• Assess founder mosaicism and germline transmission by breeding selected founders and genotyping F1 offspring, because somatic genotyping alone may not fully establish germline genotype[5][7].
Troubleshooting
Problem: Low or variable editing among loci.
• Possible Cause: Delivery format, locus-specific guide performance, and reagent format can affect editing efficiency.• Literature-supported Solution: Use validated guide design, compare RNP-based delivery with mRNA-based formats where appropriate, and confirm edits by sequencing rather than relying on a single screening assay[3][4][5][7].
Problem: Poor embryo survival after delivery.
• Possible Cause: Physical damage from microinjection or embryo-compromising pretreatment can reduce embryo development.• Literature-supported Solution: Consider electroporation of intact zygotes with RNP-based reagents, because studies reported reduced physical damage or improved embryo development compared with conventional pronuclear injection or zona-weakening workflows[4][6].
Problem: Mosaic founders.
• Possible Cause: Editing after the first genome replication can produce embryos with multiple alleles.• Literature-supported Solution: Deliver Cas9 protein/sgRNA RNPs at an early pronuclear zygote stage when feasible, because early RNP electroporation was reported to generate non-mosaic mouse mutants in the cited study[7].
Problem: Intended HDR allele is not detected.
• Possible Cause: HDR efficiency depends on donor design, target locus, delivery method, and whether the donor reaches the embryo nucleus.• Literature-supported Solution: Use ssODN donors for point mutations or small insertions when supported by the selected method, and verify HDR by sequence-level genotyping rather than assuming donor incorporation from survival or cleavage alone[3][5][6][8][9].
Referencias:
- [1]. Wang H, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910-918. [Content Brief]
- [2]. Horii T, Arai Y, Yamazaki M, Morita S, Kimura M, Itoh M, et al. Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering. Sci Rep. 2014;4:4513. [Content Brief]
- [3]. Qin W, Dion SL, Kutny PM, Zhang Y, Cheng AW, Jillette NL, et al. Efficient CRISPR/Cas9-mediated genome editing in mice by zygote electroporation of nuclease. Genetics. 2015;200(2):423-430. [Content Brief]
- [4]. Chen S, et al. Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J Biol Chem. 2016;291(28):14457-14467. [Content Brief]
- [5]. Teixeira M, Py BF, Bosc C, Laubreton D, Moutin MJ, Marvel J, et al. Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing. Sci Rep. 2018;8(1):474. [Content Brief]
- [6]. Tröder SE, et al. An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes. PLoS One. 2018;13(5):e0196891. [Content Brief]
- [7]. Hashimoto M, et al. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev Biol. 2016;418(1):1-9. [Content Brief]
- [8]. Aida T, Chiyo K, Usami T, Ishikubo H, Imahashi R, Wada Y, et al. Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biol. 2015;16:87. [Content Brief]
- [9]. Chu VT, Weber T, Graf R, Sommermann T, Petsch K, Sack U, et al. Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes. BMC Biotechnol. 2016;16:4. [Content Brief]
- [10]. Wefers B, et al. Gene editing in mouse zygotes using the CRISPR/Cas9 system. Methods. 2017;121-122:55-67. [Content Brief]
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