Precise genome editing using CRISPR typically requires delivery of guide RNAs, Cas9 endonuclease, and DNA repair templates. Both microinjection and electroporation effectively deliver these components into mouse zygotes provided the DNA template is an oligonucleotide of only a few hundred base pairs. However, electroporation completely fails with longer double-stranded DNAs leaving microinjection as the only delivery option. Here, we overcome this limitation by first injecting all CRISPR components, including long plasmid-sized DNA templates, into the sub-zona pellucida space. There they are retained, supporting subsequent electroporation. We show that this simple and well-tolerated method achieves intracellular reagent concentrations sufficient to effect precise gene edits.
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References
1.
YangH, WangH, ShivalilaCS, et al.One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell, 2013; 154:1370–1379. DOI: 10.1016/j.cell.2013.08.022.
2.
LiD, QiuZ, ShaoY, et al.Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol, 2013; 31:681–683. DOI: 10.1038/nbt.2661.
3.
WangW, KutnyPM, ByersSL, et al.Delivery of Cas9 protein into mouse zygotes through a series of electroporation dramatically increased the efficiency of model creation. J Genet Genom, 2016. DOI: 10.1016/j.jgg.2016.02.004.
4.
WangW, ZhangY, WangH. Generating mouse models using zygote electroporation of nucleases (ZEN) technology with high efficiency and throughput. Methods Mol Biol, 2017; 1605:219–230. DOI: 10.1007/978-1-4939-6988-3_15.
5.
ChenS, LeeB, LeeAY, et al.Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J Biol Chem, 2016; 29:14457–14467. DOI: 10.1074/jbc.M116.733154.
6.
KanekoT. Genome editing in mouse and rat by electroporation. Methods Mol Biol, 2017; 1630:81–89. DOI: 10.1007/978-1-4939-7128-2_7.
7.
PetersonA. CRISPR: express delivery to any DNA address. Oral Dis, 2017; 23:5–11. DOI: 10.1111/odi.12487.
8.
GrabarekJB, PlusaB, GloverDM, et al.Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation. Genesis, 2002; 32:269–276.
9.
PengH, WuY, ZhangY. Efficient delivery of DNA and morpholinos into mouse preimplantation embryos by electroporation. PLoS One, 2012; 7:e4374–8. DOI: 10.1371/journal.pone.0043748.
10.
TurnerK, HorobinRW. Permeability of the mouse zona pellucida: a structure-staining-correlation model using coloured probes. J Reprod Fertil, 1997; 111:259–265.
11.
NovoS, BarriosL, IbanezE, et al.The zona pellucida porosity: three-dimensional reconstruction of four types of mouse oocyte zona pellucida using a dual beam microscope. Microsc Microanal, 2012; 18:1442–1449. DOI: 10.1017/s1431927612013487.
12.
LiuC, WangL, LiW, et al.Highly efficient generation of transgenic sheep by lentivirus accompanying the alteration of methylation status. PLoS One, 2013; 8:e5461–4. DOI: 10.1371/journal.pone.0054614.
13.
HashimotoM, TakemotoT. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Sci Rep, 2015; 5:1131–5. DOI: 10.1038/srep11315.
14.
HashimotoM, YamashitaY, TakemotoT. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev Biol, 2016; 418:1–9. DOI: 10.1016/j.ydbio.2016.07.017.
15.
BronsonRA, McLarenA. Transfer to the mouse oviduct of eggs with and without the zona pellucida. J Reprod Fertil, 1970; 22:129–137.
16.
McLarenA. Transfer of zona-free mouse eggs to uterine foster mothers. J Reprod Fertil, 1969; 19:341–346.
17.
NemecLA, SkowLC, GoyJM, et al.Introduction of DNA into murine embryos by electroporation. Theriogenology, 1989; 31:23–3. DOI: 10.1016/0093-691X(89)90641-9.
18.
YurchenkoE, FriedmanH, HayV, et al.Ubiquitous expression of mRFP-1 in vivo by site-directed transgenesis. Transgen Res, 2007; 16:29–40. DOI: 10.1007/s11248-006-9030-6.
19.
SakuraiT, WatanabeS, KamiyoshiA, et al.A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice. BMC Biotechnol, 2014; 14:6–9. DOI: 10.1186/1472-6750-14-69.
20.
ThorvaldsdóttirH, RobinsonJT, MesirovJP. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform, 2013; 14:178–192. DOI: 10.1093/bib/bbs017.
LoCA, KaysI, EmranF, et al.Quantification of protein levels in single living cells. Cell Rep, 2015; 13:2634–2644. DOI: 10.1016/j.celrep.2015.11.048.
23.
SellensMH, JenkinsonEJ. Permeability of the mouse zona pellucida to immunoglobulin. J Reprod Fertil, 1975; 42:153–157.
24.
BeeL, FabrisS, CherubiniR, et al.The efficiency of homologous recombination and non-homologous end joining systems in repairing double-strand breaks during cell cycle progression. PLoS One, 2013; 8:e6906–1. DOI: 10.1371/journal.pone.0069061.
25.
BetermierM, BertrandP, LopezBS. Is non-homologous end-joining really an inherently error-prone process?. PLoS Genet, 2014; 10:e100408–6. DOI: 10.1371/journal.pgen.1004086.
26.
YenST, ZhangM, DengJM, et al.Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev Biol, 2014; 393:3–9. DOI: 10.1016/j.ydbio.2014.06.017.
27.
YasueA, KonoH, HabutaM, et al.Relationship between somatic mosaicism of Pax6 mutation and variable developmental eye abnormalities-an analysis of CRISPR genome-edited mouse embryos. Sci Rep, 2017; 7:5–3. DOI: 10.1038/s41598-017-00088-w.
28.
MikuniT, NishiyamaJ, SunY, et al.High-throughput, high-resolution mapping of protein localization in mammalian brain by in vivo genome editing. Cell, 2016; 165:1803–1817. DOI: 10.1016/j.cell.2016.04.044.
29.
BurgerA, LindsayH, FelkerA, et al.Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes. Development, 2016; 143:2025–2037. DOI: 10.1242/dev.134809.
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