通过受精卵的CRISPR电穿孔对小鼠进行高效基因组编辑
Summary
在这里，我们描述了一种用于高效生成转基因小鼠的简单技术，称为CRISPR RNP受精卵电穿孔（CRISPR-EZ）。该方法通过电穿孔将编辑试剂以接近100%的效率输送到胚胎中。该方案对哺乳动物胚胎中的点突变、小基因组插入和缺失有效。
Abstract
凭借卓越的效率、准确性和易用性，CRISPR/Cas9系统显著改善了细胞培养和实验动物实验中的基因组编辑。在生成动物模型时，受精卵电穿孔可提供更高的效率、简单性、成本和通量，作为显微注射金标准方法的替代方案。电穿孔也更温和，具有更高的活力，并且可靠地将Cas9/单向导RNA（sgRNA）核糖核蛋白（RNP）递送到常见实验室小鼠品系（例如C57BL / 6J和C57BL / 6N）的受精卵中，接近100%的递送效率。该技术可实现插入/缺失（插入缺失）突变、点突变、整个基因或外显子的缺失，以及 100-200 bp 范围内的小插入以插入 LoxP 或短标签，如 FLAG、HA 或 V5。在不断改进的同时，我们在这里展示了CRISPR-EZ的当前状态，其中包括通过 体外 转录，胚胎处理，RNP组装，电穿孔和植入前胚胎的基因分型产生sgRNA。具有最少操作胚胎经验的研究生水平的研究人员可以使用该协议在不到 1 周的时间内获得经过基因编辑的胚胎。在这里，我们提供了一种简单，低成本，高效，高容量的方法，可用于小鼠胚胎。
Introduction
自CRISPR编辑1，2，3出现以来，活小鼠的基因组编辑已经大大简化，并且变得容易获得且更实惠。最初的动物编辑尝试使用显微注射将CRISPR Cas9 mRNA / sgRNA递送到前核阶段胚胎中4，5，6。虽然显微注射非常有效，但完全掌握它所需的练习量可能不适合受训者和学生，并且还需要昂贵的设备，而资金有限的实验室无法负担。显微注射通常由转基因设施的专家技术人员进行，其时间表和服务价格对许多研究人员来说是限速的。一种更容易获得的方法是电穿孔，它已被证明对于将CRISPR Cas9 mRNA / sgRNA递送到前核阶段胚胎中非常有效7。CRISPR基因组编辑和递送策略的进一步改进表明，已经与sgRNA结合的预组装RNP可能是减少嵌合体的有效手段8。
开发和使用该方案背后的基本原理是绕过与显微注射相关的许多限制和障碍。顾名思义，一种简单、内部且经济高效的方法可以快速确定未经测试的 sgRNA 设计是否值得在显微进样实验中使用，这将是一个非常方便的首过质量控制步骤（图 1）。虽然这种方法不能取代显微注射用于更复杂的策略，例如为基于重组的结果引入长供体DNA序列，但它是不太复杂的策略的理想选择，如小的缺失或插入以及标记基因。这种方法适用于具有基本胚胎操作技能的研究人员，他们有简单的编辑需求，希望在植入前发育的时间范围内测试他们的假设，或者更喜欢在安排与显微注射专家的预约之前测试胚胎中的sgRNA。在这里，编辑试剂通过电穿孔（一系列电脉冲） 以 Cas9 / sgRNA RNP的形式瞬时递送到前核阶段胚胎中，以最大限度地提高效率，同时减少嵌合体8。使用胚胎基因分型方法，编辑结果可在大约 1 周内获得9，从而以显着降低的成本减少了对各种显微注射应用的需求。
该方法的有效性在原核胚胎阶段达到峰值，此时胚胎尚未融合母本和父本原核或进入S期（图2）。超排卵用于最大化受精卵的数量，但同时产生原核受精卵和未受精卵。健康的受精卵也可以在电穿孔前预先选择，以提高整体效率。由于其他电穿孔方案已经有效地编辑了受精卵，而无需包括类似的步骤7，10，11，12，13，14，15，因此该协议的可选步骤是透明带（ZP）的轻微侵蚀。ZP 是一种糖蛋白层，有助于精子结合、顶体反应和核前期胚胎周围的受精。根据我们的经验，我们发现对ZP的温和酸基侵蚀提供了可靠的Cas9 RNP电穿孔递送，对活性的影响很小。
我们已经观察到，在C57BL / 6J和C57BL /6N 9，16等研究中常用的小鼠品系中，通过电穿孔的RNP递送率高达100%。独立小组还开发了基于电穿孔的程序，其效率大于或匹配显微注射11，12，13，14，15，17，电穿孔方案在大鼠18，19，猪20，21，22和牛23中运行良好，因此我们建议读者比较协议以找到最适合其实验和设备需求的条件。这里描述的系统使用常见的材料和设备，只需要基本的胚胎操作技能。该技术对一系列编辑策略有效，使该方法被研究界广泛使用。
设计理想的小向导RNA（sgRNA）对于高效编辑至关重要。我们建议直接在小鼠胚胎中为每个靶位点筛选两到三种sgRNA策略，尤其是在需要生成小鼠系的情况下。一旦设计出来，建议使用体外转录（IVT）等无克隆方法来产生高质量的sgRNA3。将RNP和sgRNA与30-50个处理过的前核阶段胚胎混合，并暴露于一系列电脉冲中，首先暂时透化ZP和细胞膜，随后的脉冲保持孔隙打开并通过受精卵24对RNP进行电泳。优化后，我们发现在30 V下对大体胚胎（~50）的6个3 ms脉冲最适合编辑有效性和活力，提供高效的Cas9 / sgRNA RNP递送9，16，25。可以使用 CRISPR 编辑中常见的各种验证策略来确认单个小鼠桑葚中的编辑事件，例如限制性片段长度多态性 （RFLP）、T7 核酸内切酶消化和感兴趣区域的 Sanger 测序26。
目前的方法最适合简单的编辑方案（图3），例如插入/缺失（插入缺失），外显子大小的500-2000 bp缺失，以及点突变和小插入的传递，例如C或N端标签（例如，FLAG，HA或V5）9，16，27。复杂基因组编辑的潜力，如大量插入荧光标签或条件等位基因，仍然不确定，并且是即将进行的改进的当前重点。
该方法易于掌握，可用于在1周内 快速测试培养小鼠胚胎中的sgRNA（图1）。这项工作中提出的是一个六步方案，其中包括1）sgRNA设计;2）sgRNA合成;3）超排卵和交配;4）胚胎培养、采集和加工;5）RNP组装和电穿孔;6）胚胎培养和基因分型。提供有关所有所用材料的信息（材料表）。作为阳性对照，编辑 酪氨酸酶（Tyr）位点9，16 的试剂已包含在 补充表1中。
Protocol
Representative Results
Discussion
这里介绍的是一种简单高效的小鼠基因组编辑技术。电穿孔可用于在1-2周内产生修饰的胚胎（图1），并且可以在6周内产生经过编辑的小鼠9。与同时期开发的基于电穿孔的RNPs7，10，11，12，13，14，15，17，<su…
Divulgations
The authors have nothing to disclose.
Acknowledgements
A.J.M.创造了导致CRISPR-EZ发展的原始概念并产生了这些数字。C.K.D.为当前手稿汇编并改编了内部和已发表的协议。A.J.M. 由 NIH （R00HD096108） 提供支持。
Materials
| 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 |
References
- Cong, L., et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 339 (6121), 819-823 (2013).
- Xu, H., et al. Sequence determinants of improved CRISPR sgRNA design. Genome Research. 25 (8), 1147-1157 (2015).
- Lin, S., Staahl, B., Alla, R. K., Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife. 3, 04766 (2014).
- Wang, B., et al. Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes. Biotechniques. 59 (4), 201-208 (2015).
- Yang, H., Wang, H., Jaenisch, R. Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. Nature Protocols. 9 (8), 1956-1968 (2014).
- Fujii, W., Onuma, A., Sugiura, K., Naito, K. One-step generation of phenotype-expressing triple-knockout mice with heritable mutated alleles by the CRISPR/Cas9 system. Journal of Reproduction and Development. 60 (4), 324-327 (2014).
- Kaneko, T., et al. Simple genome editing of rodent intact embryos by electroporation. PLoS One. 10 (11), 0142755 (2015).
- Mehravar, M., Shirazi, A., Nazari, M., Banan, M. Mosaicism in CRISPR/Cas9-mediated genome editing. Biologie du développement. 445 (2), 156-162 (2019).
- Modzelewski, A. J., et al. Efficient mouse genome engineering by CRISPR-EZ technology. Nature Protocols. 13 (6), 1253-1274 (2018).
- Gurumurthy, C. B., et al. Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD. Nature Protocols. 14 (8), 2452-2482 (2019).
- Hur, J. K., et al. Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nature Biotechnology. 34 (8), 807-808 (2016).
- Tröder, S. E., et al. An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes. PLoS One. 13 (5), 0196891 (2018).
- Qin, W., et al. Efficient CRISPR/cas9-mediated genome editing in mice by zygote electroporation of nuclease. Génétique. 200 (2), 423-430 (2015).
- Hashimoto, M., Yamashita, Y., Takemoto, T. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Biologie du développement. 418 (1), 1-9 (2016).
- Teixeira, M., et al. Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing. Scientific Reports. 8, 474 (2018).
- Chen, S., Lee, B., Lee, A. Y. -. F., Modzelewski, A. J., He, L. Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. Journal of Biological Chemistry. 291 (28), 14457-14467 (2016).
- Wang, W., et al. Delivery of Cas9 protein into mouse zygotes through a series of electroporation dramatically increased the efficiency of model creation. Journal of Genetics and Genomics. 43 (5), 319-327 (2016).
- Mizuno, N., et al. Intra-embryo gene cassette knockin by CRISPR/Cas9-mediated genome editing with adeno-associated viral vector. iScience. 9, 286-297 (2018).
- Remy, S., et al. Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation. Scientific Reports. 7, 16554 (2017).
- Tanihara, F., et al. Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation. Animal Science Journal. 90 (1), 55-61 (2019).
- Zhou, X., Guo, H., Chen, K., Cheng, H., Zhou, R. Identification, chromosomal mapping and conserved synteny of porcine Argonaute family of genes. Genetica. 138 (7), 805-812 (2010).
- Nishio, K., et al. Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos. Reproduction in Domestic Animals. 53 (2), 313-318 (2018).
- Camargo, L. S. A., Owen, J. R., van Eenennaam, A. L., Ross, P. J. Efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos. Frontiers in Genetics. 11, 570069 (2020).
- Escoffre, J. M., et al. What is (still not) known of the mechanism by which electroporation mediates gene transfer and expression in cells and tissues. Molecular Biotechnology. 41 (3), 286-295 (2009).
- Chen, S., et al. CRISPR-READI: Efficient generation of knockin mice by CRISPR RNP electroporation and AAV donor infection. Cell Reports. 27 (13), 3780-3789 (2019).
- Sentmanat, M. F., Peters, S. T., Florian, C. P., Connelly, J. P., Pruett-Miller, S. M. A survey of validation strategies for CRISPR-Cas9 editing. Scientific Reports. 8, 888 (2018).
- Modzelewski, A. J., et al. A mouse-specific retrotransposon drives a conserved Cdk2ap1 isoform essential for development. Cell. 184 (22), 5541-5558 (2021).
- Xu, H., et al. Sequence determinants of improved CRISPR sgRNA design. Genome Research. 25 (8), 1147-1157 (2015).
- Sanson, K. R., et al. Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. Nature Communications. 9 (1), 5416 (2018).
- Okamoto, S., Amaishi, Y., Maki, I., Enoki, T., Mineno, J. Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs. Scientific Reports. 9, 14811 (2019).
- Duselis, A. R., Vrana, P. B. Retrieval of mouse oocytes. Journal of Visualized Experiments. (3), e185 (2007).
- Takahashi, G., et al. GONAD: Genome-editing via oviductal nucleic acids delivery system: A novel microinjection independent genome engineering method in mice. Scientific Reports. 5, 11406 (2015).
- Tu, Z., et al. Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos. Scientific Reports. 7, 42081 (2017).
- Aida, T., et al. Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biology. 16 (1), 87 (2015).
- Zhu, L., et al. Patterns of exon-intron architecture variation of genes in eukaryotic genomes. BMC Genomics. 10, 47 (2009).
- Quadros, R. M., et al. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biology. 18, 92 (2017).
- Gurumurthy, C. B., Saunders, T. L., Ohtsuka, M. Designing and generating a mouse model: frequently asked questions. Journal of Biomedical Research. 35 (2), 76-90 (2021).
- Nakajima, K., et al. Exome sequencing in the knockin mice generated using the CRISPR/ Cas system. Scientific Reports. 6, 34703 (2016).
- Iyer, V., et al. Off-target mutations are rare in Cas9-modified mice. Nature Methods. 12 (6), 479 (2015).
- Wang, H., et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 153 (4), 910-918 (2013).
