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Here, we describe a simple technique intended for the efficient generation of genetically modified mice called CRISPR RNP Electroporation of Zygotes (CRISPR-EZ). This method delivers editing reagents by electroporation into embryos at an efficiency approaching 100%. This protocol is effective for point mutations, small genomic insertions, and deletions in mammalian embryos.
With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal experiments. When generating animal models, the electroporation of zygotes offers higher efficiency, simplicity, cost, and throughput as an alternative to the gold standard method of microinjection. Electroporation is also gentler, with higher viability, and reliably delivers Cas9/single-guide RNA (sgRNA) ribonucleoproteins (RNPs) into the zygotes of common laboratory mouse strains (e.g., C57BL/6J and C57BL/6N) that approaches 100% delivery efficiency. This technique enables insertion/deletion (indels) mutations, point mutations, the deletion of whole genes or exons, and small insertions in the range of 100-200 bp to insert LoxP or short tags like FLAG, HA, or V5. While constantly being improved, here we present the current state of CRISPR-EZ in a protocol that includes sgRNA production through in vitro transcription, embryo processing, RNP assembly, electroporation, and the genotyping of preimplantation embryos. A graduate-level researcher with minimal experience manipulating embryos can obtain genetically edited embryos in less than 1 week using this protocol. Here, we offer a straightforward, low-cost, efficient, high-capacity method that could be used with mouse embryos.
Genome editing in live mice has been considerably simplified and has become accessible and more affordable since the emergence of CRISPR editing1,2,3. Initial animal editing attempts used microinjection to deliver CRISPR Cas9 mRNA/sgRNA into pronuclear-stage embryos4,5,6. While microinjection is quite effective, the amount of practice required to fully master it might not be appropriate for trainees and students and also requires expensive equipment that a modestly funded lab is unable to afford. Microinjection is normally performed by expert technicians at transgenic facilities with schedules and service prices that are rate-limiting for many researchers. A more accessible approach is that of electroporation, which has been demonstrated to be quite effective for the delivery of CRISPR Cas9 mRNA/sgRNA into pronuclear-stage embryos7. Further improvements in CRISPR genome editing and delivery strategies suggested that pre-assembled RNPs already engaged with sgRNAs may be an effective means to reduce mosaicism8.
The rationale behind the development and use of this protocol was to bypass many of the limitations and obstacles associated with microinjection. As the name implies, an easy, in-house, and cost-effective method that could quickly determine whether untested sgRNA designs would be worthwhile using during a microinjection experiment would be a very convenient first pass quality control step (Figure 1). While this method cannot replace microinjection for more complex strategies, like introducing long donor DNA sequences for recombination-based outcomes, it is ideal for less complex strategies like small deletions or insertions and tagging genes. This method is appropriate for researchers with basic embryo manipulation skills who have simple editing needs, would like to test their hypothesis within the timeframe of preimplantation development, or prefer to test sgRNAs in embryos before scheduling an appointment with a microinjection specialist. Here, editing reagents are transiently delivered into pronuclear-stage embryos as Cas9/sgRNA RNPs via electroporation (a series of electrical pulses) to maximize efficiency while decreasing mosaicism8. Using an embryo genotyping method, editing results are available in approximately 1 week9, thus reducing the need for various microinjection applications at a significantly reduced cost.
This method's effectiveness peaks at the pronuclear embryo stage, when the embryo has not yet fused the maternal and paternal pronuclei or entered S-phase (Figure 2). Superovulation is used to maximize the number of zygotes but produces both pronuclear zygotes and unfertilized eggs. Healthy zygotes can also be pre-selected before electroporation to increase the overall efficiency. As other electroporation protocols have efficiently edited zygotes without the need to include a similar step7,10,11,12,13,14,15, an optional step of this protocol is the slight erosion of the zona pellucida (ZP). The ZP is a glycoprotein layer that aids spermatozoa binding, acrosome response, and fertilization surrounding pronuclear-stage embryos. In our experience, we found that a gentle acid-based erosion of the ZP provides reliable Cas9 RNP electroporation delivery with only a marginal impact on viability.
We have observed RNP delivery rates of up to 100% efficiency via electroporation in mouse strains that are commonly used in research like C57BL/6J and C57BL/6N9,16. Independent groups have also developed electroporation-based procedures with efficiencies greater than or matching microinjection11,12,13,14,15,17, with electroporation protocols functioning well in rat18,19, pig20,21,22 and cow23, so we suggest that readers compare the protocols to find the conditions that best suit their experimental and equipment needs. The system described here uses common materials and equipment, requiring only basic embryo manipulation skills. This technique is effective for a range of editing strategies, making this method broadly accessible to the research community.
Designing ideal small guide RNAs (sgRNAs) is essential for efficient editing. We recommend screening two to three sgRNA strategies per target site directly in mouse embryos, especially if mouse line generation is desired. Once designed, cloning-free methods like in vitro transcription (IVT) to produce high-quality sgRNAs are recommended3. The RNPs and sgRNAs are mixed with 30-50 processed pronuclear-stage embryos and exposed to a series of electrical pulses to first temporarily permeabilize the ZP and cell membrane, with subsequent pulses to keep the pores open and electrophorese the RNPs through the zygote24. After optimization, we found that six 3 ms pulses at 30 V for bulk embryos (~50) were optimal for editing effectiveness and viability, providing highly efficient Cas9/sgRNA RNP delivery9,16,25. Editing events in individual mouse morula can be confirmed using a variety of validation strategies common for CRISPR editing, such as restriction fragment length polymorphism (RFLP), T7 endonuclease digestion, and Sanger sequencing of the region of interest26.
The current method is most appropriate for simple editing schemes (Figure 3), such as insertion/deletions (indels), exon-sized deletions on the order of 500-2000 bp, and the delivery of point mutations and small insertions such as C- or N-Terminal tags (e.g., FLAG, HA, or V5)9,16,27. The potential for complex genome editing, like large insertions of fluorescent tags or conditional alleles, remains uncertain and is the present focus of upcoming improvements.
This method is easily mastered and can be used to quickly test sgRNAs in cultured mouse embryos in 1 week9 (Figure 1). Presented in this work is a six-step protocol, which includes 1) sgRNA design; 2) sgRNA synthesis; 3) superovulation and mating; 4) embryo culture, collection, and processing; 5) RNP assembly and electroporation; 6) embryo culture and genotyping. Information about all the materials used is provided (Table of Materials). As a positive control, reagents to edit the Tyrosinase (Tyr) locus9,16 have been included in the Supplementary Table 1.
All animal care and use throughout this protocol adhered to Animal Welfare Act policies the ILAR Guide for Care and Use of Laboratory Animals and followed guidelines from the AVMA for euthanasia and the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) guidelines and policies. The animal care and use protocol was reviewed and approved by the University of Pennsylvania IACUC for this project. As a matter of compliance and caution, please seek out all necessary authorizations prior to attempting this protocol.
1. sgRNA and optional donor oligo design
2. sgRNA synthesis
3. Superovulation
4. Embryo collection and processing
5. RNP assembly and electroporation
6. Embryo culture and genotyping
This method generates more than 100 µg of sgRNA (20 µL at >6,000 ng/L concentration) for efficient Cas9/sgRNA RNP assembly. The routine superovulation method described here typically produces 10-20 viable embryos per plugged female. Due to handling errors and typical losses associated with embryo manipulation, an expected 80% of embryos are fertilized, viable, and in excellent condition after electroporation. To aid researchers in executing a successful experiment, we have provided an example strategy to ta...
Presented here is a straightforward and highly efficient mouse genome editing technology. Electroporation can be used to generate modified embryos in 1-2 weeks (Figure 1) and can produce edited mice within 6 weeks9. Compared to contemporaneously developed electroporation-based protocols that deliver RNPs7,10,11,12,
There are no relevant financial declarations by the authors.
A.J.M. created the original concept that led to the development of CRISPR-EZ and produced the figures. C.K.D. compiled and adapted the internal and published protocols for this current manuscript. A.J.M. is supported by NIH (R00HD096108).
Name | Company | Catalog Number | Comments |
0.1-cm-gap electroporation cuvette | Bio-Rad | cat. no. 1652089 | Electroporation |
26-G, 1/2-inch needle | BD | cat. no. 305111 | Superovulation |
3–8-month-old male mice and 3- to 5-week-old female mice | JAX | cat. no. 000664 | Superovulation |
35-mm Tissue culture dish | Greiner Bio-One, | cat. no. 627-160 | Embryo Culture |
60-mm Tissue culture dish | Greiner Bio-One, | cat. no. 628-160 | Embryo Processing |
6x loading dye | Thermo Fisher Scientific | cat. no. R0611 | sgRNA Synthesis and Genotyping |
Acidic Tyrode's (AT) solution, embryo culture grade | Sigma-Aldrich, | cat. no. T1788 | Embryo Processing |
BSA, embryo culture grade | Sigma-Aldrich | cat. no. A3311 | Embryo Processing and Culture |
Cas9 protein | Alt-R S.p. Cas9 nuclease 3NLS | cat. no. 1074181 | Electroporation |
DNase I, RNase-free | New England BioLabs, | cat. no. M0303 | sgRNA Synthesis |
DPBS(calcium and magnesium free) | Gibco | cat. no. 14190-144 | Embryo Processing |
EcoRI | NEB | cat. no. R3101S | Genotyping |
EDTA, anhydrous | Sigma-Aldrich | cat. no. EDS-100G | RNP Buffer |
Ethanol | Koptec | cat. no. V1016 | sgRNA Synthesis |
Gelatin (powder) type B, laboratory grade | Fisher, | cat. no. G7-500 | Lysis Buffer |
Glycerol, molecular-biology grade | Fisher | cat. no. BP229 | RNP Buffer |
Taq Polymerase | Promega | cat. no. M712 | Genotyping |
HEPES, cell culture grade | Sigma-Aldrich | cat. no. H4034 | RNP Buffer |
HinfI (10,000 U/mL) | NEB | cat. no. R0155S | Genotyping |
HiScribe T7 High Yield RNA Synthesis Kit | New England BioLabs, | cat. no. E2040 | sgRNA Synthesis |
Human chorion gonadotropin, lyophilized (hCG) | Millipore | cat. no. 230734 | Superovulation |
Hyaluronidase/M2 | Millipore | cat. no. MR-051-F | Embryo Processing |
KSOMaa Evolve medium (potassium-supplemented simplex-optimized medium plus amino acids) | Zenith Biotech | cat. no. ZEKS-050 | Embryo Culture |
LE agarose, analytical grade | BioExpress | cat. no. E-3120-500 | sgRNA Synthesis and Genotyping |
M2 medium | Zenith Biotech | cat. no. ZFM2-050 | Embryo Processing |
Magnesium chloride, anhydrous (MgCl2) | Sigma-Aldrich | cat. no. M8266 | RNP and Lysis Buffer |
Mineral Oil | Millipore | cat. no. ES-005C | Embryo Culture |
Nonidet P-40,substitute (NP-40) | Sigma-Aldrich | cat. no. 74385 | Lysis Buffer |
Nuclease-free water, molecular-biology grade | Ambion | cat. no. AM9937 | sgRNA Synthesis and Genotyping |
Oligos for sgRNA synthesis, donor oligo and PCR primers for genotyping | Integrated DNA Technologies | custom orders | sgRNA Design |
Reduced serum medium | Thermo Fisher Scientific | cat. no. 31985062 | Embryo Culture |
High-fidelity DNA polymerase | New England BioLabs, | cat. no. M0530 | sgRNA Synthesis |
Potassium chloridemolecular-biology grade (KCl) | Sigma-Aldrich | cat. no. P9333 | RNP and Lysis Buffer |
Pregnant mare serum gonadotropin lyophilizd ((PMSG) | ProspecBio | cat. no. HOR-272 | Superovulation |
Proteinase K, molecular-biology grade | Fisher | cat. no. BP1700-100 | Lysis Buffer |
RNase-free 1.5-mL microcentrifuge tube | VWR | cat. no. 20170-333 | sgRNA Synthesis and Genotyping |
RNase-free eight-well PCR strip tubes | VWR | cat. no. 82006-606 | sgRNA Synthesis and Genotyping |
Magnetic purification beads | GE Healthcare | cat. no. 65152105050250 | sgRNA Synthesis |
Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) | Sigma-Aldrich | cat. no. C4706 | RNP Buffer |
Tris-HCl solution, pH 8.5 molecular-biology grade | Teknova | cat. no. T1085 | Lysis Buffer |
Tween 20 molecular-biology grade | Sigma-Aldrich | cat. no. P7949-500 | Lysis Buffer |
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