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Efficient mouse genome engineering by CRISPR-EZ technology

Abstract

CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. Here, we provide an optimized protocol covering sgRNA synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher with basic embryo-manipulation skills can obtain genetically modified mice in 6 weeks. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species.

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Figure 1: Overview of CRISPR-EZ technology and workflow.
Figure 2: Optimization of CRISPR-EZ conditions for editing efficiency and embryo viability.
Figure 3: Comparing editing efficiency and viability of CRISPR-EZ and microinjection.
Figure 4: Diagram illustrating a cloning-free strategy for sgRNA synthesis.
Figure 5: Overview of key superovulation and zygote collection procedures.
Figure 6: An overview of key zygote-processing stages.
Figure 7: An overview of key procedures for oviduct transfer.

References

  1. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Li, D. et al. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat. Biotechnol. 31, 681–683 (2013).

    CAS  PubMed  Google Scholar 

  6. Yasue, A. et al. Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems. Sci. Rep. 4, 5705 (2015).

    Google Scholar 

  7. Fujii, W., Kawasaki, K., Sugiura, K. & Naito, K. Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Res. 41, e187 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Yang, H. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Hashimoto, M., Yamashita, Y. & Takemoto, T. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev. Biol. 418, 1–9 (2016).

    CAS  PubMed  Google Scholar 

  10. Tu, Z. et al. Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos. Sci. Rep. 7, 42081 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Aida, T. et al. Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biol. 16, 87 (2015).

    PubMed  PubMed Central  Google Scholar 

  12. Jung, C.J. et al. Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein. Transgenic Res. 26, 263–277 (2017).

    CAS  PubMed  Google Scholar 

  13. Liang, X. et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J. Biotechnol. 208, 44–53 (2015).

    CAS  PubMed  Google Scholar 

  14. Skarnes, W.C. Is mouse embryonic stem cell technology obsolete? Genome Biol. 16, 109 (2015).

    PubMed  PubMed Central  Google Scholar 

  15. Sung, Y.H. et al. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res. 24, 125–131 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Chen, S., Lee, B., Lee, A.Y.-F., Modzelewski, A.J. & He, L. Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J. Biol. Chem. 291, 14457–14467 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Wang, W. et al. Delivery of Cas9 protein into mouse zygotes through a series of electroporation dramatically increases the efficiency of model creation. J. Genet. Genomics 43, 319–327 (2016).

    PubMed  PubMed Central  Google Scholar 

  18. Hur, J.K. et al. Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat. Biotechnol. 34, 807–808 (2016).

    CAS  PubMed  Google Scholar 

  19. Qin, W. et al. Efficient CRISPR/Cas9-mediated genome editing in mice by zygote electroporation of nuclease. Genetics 200, 423–430 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Hashimoto, M. & Takemoto, T. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Sci. Rep. 5, 11315 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Takahashi, G. et al. GONAD: genome-editing via oviductal nucleic acids delivery system: a novel microinjection independent genome engineering method in mice. Sci. Rep. 5, 11406 (2015).

    PubMed  PubMed Central  Google Scholar 

  22. Kaneko, T., Mashimo, T. & Zhang, Y. et al. Simple genome editing of rodent intact embryos by electroporation. PLoS One 10, e0142755 (2015).

    PubMed  PubMed Central  Google Scholar 

  23. Zuris, J.A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).

    CAS  PubMed  Google Scholar 

  24. Singh, P., Schimenti, J.C. & Bolcun-Filas, E. A mouse geneticist's practical guide to CRISPR applications. Genetics 199, 1–15 (2015).

    CAS  PubMed  Google Scholar 

  25. Tanihara, F. et al. Somatic cell reprogramming-free generation of genetically modified pigs. Sci. Adv. 2, e1600803 (2016).

    PubMed  PubMed Central  Google Scholar 

  26. Nakagawa, Y. et al. Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice. Biol. Open 5, 1142–1148 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Park, K.-E. et al. Targeted gene knock-in by CRISPR/Cas ribonucleoproteins in porcine zygotes. Sci. Rep. 7, 42458 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Liang, P. et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell 6, 363–372 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. DeWitt, M.A., Corn, J.E. & Carroll, D. Genome editing via delivery of Cas9 ribonucleoprotein. Methods 121–122, 9–15 (2017).

    PubMed  PubMed Central  Google Scholar 

  30. Doetschman, T. et al. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330, 576–578 (1987).

    CAS  PubMed  Google Scholar 

  31. Doetschman, T., Maedat, N. & Smithiestt, O. Targeted mutation of the Hprt gene in mouse embryonic stem cells (gene targeting/embryonic stem cells/homologous recombination/polymerase chain reaction amplification). Genetics 85, 8583–8587 (1988).

    CAS  Google Scholar 

  32. 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 Biol. 18, 92 (2017).

    PubMed  PubMed Central  Google Scholar 

  33. Howe, C.C. & Solter, D. Cytoplasmic and nuclear protein synthesis in preimplantation mouse embryos. J. Embryol. Exp. Morphol. 52, 209–225 (1979).

    CAS  PubMed  Google Scholar 

  34. Cullen, B.R., Emigholz, K. & Monahan, J.J. Protein patterns of early mouse embryos during development. Differentiation 17, 151–160 (1980).

    CAS  PubMed  Google Scholar 

  35. Wang, W. et al. Delivery of Cas9 protein into mouse zygotes through a series of electroporation dramatically increases the efficiency of model creation. J. Genet. Genomics 43, 319–327 (2016).

    PubMed  PubMed Central  Google Scholar 

  36. Nagy, A., Gertenstein, M., Vintersten, K. & Behringer, R. Manipulating the Mouse Embryo: A Laboratory Manual 3rd edn. (Cold Spring Harbor Laboratory Press, 2003).

  37. Zhu, L. et al. Patterns of exon-intron architecture variation of genes in eukaryotic genomes. BMC Genomics 10, 47 (2009).

    PubMed  PubMed Central  Google Scholar 

  38. Schaefer, K.A. et al. Unexpected mutations after CRISPR–Cas9 editing in vivo. Nat. Methods 14, 547–548 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Nakajima, K. et al. Exome sequencing in the knockin mice generated using the CRISPR/Cas system. Sci. Rep. 6, 34703 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Iyer, V. et al. Off-target mutations are rare in Cas9-modified mice. Nat. Methods 12, 479–479 (2015).

    CAS  PubMed  Google Scholar 

  41. Slaymaker, I.M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).

    CAS  PubMed  Google Scholar 

  42. Kleinstiver, B.P. et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Moreno-Mateos, M.A. et al. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat. Methods 12, 982–988 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Xu, H. et al. Sequence determinants of improved CRISPR sgRNA design. Genome Res. 25, 1147–1157 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Horlbeck, M.A. et al. Nucleosomes impede Cas9 access to DNA in vivo and in vitro. Elife 5, e12677 (2016).

    PubMed  PubMed Central  Google Scholar 

  46. 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, e04766 (2014).

    PubMed  PubMed Central  Google Scholar 

  47. Godine, J.E., Chin, W.W. & Habener, J.F. alpha Subunit of rat pituitary glycoprotein hormones. Primary structure of the precursor determined from the nucleotide sequence of cloned cDNAs. J. Biol. Chem. 257, 8368–8371 (1982).

    CAS  PubMed  Google Scholar 

  48. Wassarman, P.M. Mammalian fertilization: molecular aspects of gamete adhesion, exocytosis, and fusion. Cell 96, 175–183 (1999).

    CAS  PubMed  Google Scholar 

  49. Malter, H.E. & Cohen, J. Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos. Gamete Res. 24, 67–80 (1989).

    CAS  PubMed  Google Scholar 

  50. Saito, T. In vivo electroporation in the embryonic mouse central nervous system. Nat. Protoc. 1, 1552–1558 (2006).

    CAS  PubMed  Google Scholar 

  51. 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. Mol. Biotechnol. 41, 286–295 (2009).

    CAS  PubMed  Google Scholar 

  52. Urnov, F.D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).

    CAS  PubMed  Google Scholar 

  53. Kim, H.J., Lee, H.J., Kim, H., Cho, S.W. & Kim, J.-S. Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Res. 19, 1279–1288 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Morbeck, D.E. & Leonard, P.H. in Embryo Culture 325–331 Humana Press, (2012).

  55. Wang, Z. & Storm, D.R. Extraction of DNA from mouse tails. Biotechniques 41, 410 412 (2006).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank A. Lee and B. Lee for technical assistance. We thank C. Di Biagio for help in making the protocol video. We thank C. Jeans and the University of California, Berkeley (UC Berkeley) California Institute for Quantitative Biosciences (QB3) Macrolab for purified Cas9 protein used during testing. We thank J. Doudna, D. Carrol, and J. Corn for stimulating discussions, and N. Anchell, P. Dao, K. Jager, and D. Young for zygote collection, electroporation, and embryo transfer. We thank K.N. Grimsrud for veterinary oversight. L.H. was supported by a Howard Hughes Medical Institute (HHMI) Faculty Scholar award, a Bakar Fellow award at UC Berkeley, several grants from the National Institutes of Health (NIH; R01GM114414, R01CA139067, 2R01CA139067, 1R21HD088885 and R21HD088885), and a research scholar award from the American Cancer Society. A.J.M. was supported by a F32 postdoctoral fellowship from the NIH (CA192636-03). S.C. was supported by a CCRC predoctoral fellowship and a Siebel predoctoral fellowship. J.A.W. and K.C.K.L. acknowledge support from two NIH grants (UM1 OD023221 and U42 OD011175) and a grant from the American College of Laboratory Animal Medicine.

Author information

Authors and Affiliations

Authors

Contributions

A.J.M. conceived the original idea that led to the development of the CRISPR-EZ technology, drafted the manuscript and methods, analyzed the raw experimental data, and produced all figures and tables. S.C. and A.J.M. trained the staff at the Mouse Biology Program (MBP) at UC Davis to perform CRISPR-EZ experiments, optimized electroporation conditions and produced a video for all experimental procedures. S.C. conceived of the Tyr editing scheme for optimizing the CRISPR-EZ protocol, and drafted and revised the manuscript. B.J.W. contributed to sgRNA design, RNP preparation, genotype screening, data analysis, and manuscript preparation. J.A.W. and K.C.K.L. contributed to the study design, collaboration setup, and paper revision. L.H. contributed to the experimental design, initiated and established the collaboration with MBP at UC Davis, and worked closely with A.J.M. and S.C. to interpret the data and to draft and revise the manuscript.

Corresponding authors

Correspondence to K C Kent Lloyd, Joshua A Wood or Lin He.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 CRISPR-EZ enables high-throughput genome editing.

(a) No appreciable differences are observed in editing efficiency (measured by mouse coat color, left) or viability (measured by live birth rate, right) when performing CRISPR-EZ using a pool of 35, 60 or 100 C57B/6J pronuclear embryos. (b) Since Tyr deficiency causes albinism in edited mice, the extent of albinism correlates the extent of editing that disrupted Tyr function. Representative image is shown for the edited mice from experiments in A, (100 Pooled Embryos). (c) The size of deletion affects editing efficiency using CRISPR-EZ. Deletions in the range of 1030-2672 bp (n=6) exhibit reduced efficiency compared to those ranging from 383-955 bp (n=16). Data are means ± SD across all genes. ** P < 0.01; *** P < 0.001; **** P < 0.0001; n.s., not significant (All P-values were calculated on a basis of an unpaired one-tailed Student’s t test.)

Supplementary Figure 2 Successful examples of targeted exon deletions by CRISPR-EZ.

Experimental design and representative genotyping results are shown for several successful CRISPR-EZ projects that aim to employ up to two pairs of sgRNAs to mediate deletion of key protein-coding exon(s) (red exons). Successfully targeted genes by CRISPR-EZ include Copb1 (a), Xpo5 (b), Kpna2 (c), Ipo7 (d), Slc25a18 (e), Mcts1 (f), Unc5cl (g) and Wdr82 (h). QIAxcel fragment analysis image of PCR genotyping results as well as chromatograms of validated sequence at the edited sites are shown accompanying each diagram. NTC, non-template control; WT, wild type.

Supplementary Figure 3 Successful examples of targeted exon deletions by CRISPR-EZ that are refractory to microinjection.

Experimental designs are shown genes in which CRISPR-EZ and Microinjection methodologies were directly tested against one another. Successfully targeted genes by CRISPR-EZ include Gsg1l (a), Fubp1 (b), Clcnkb (c), Cdnp2 (d), Ddx6 (e), Cfap57 (f), Gndp2 (g) and Sfmbt2 (h). In the case of Clcnkb, three guides were used as indicated in (c). QIAxcel fragment analysis image of PCR genotyping results as well as chromatograms of validated sequence at the edited sites are shown accompanying each diagram. Schematics without QIAxcel fragment analysis and chromatograms are pending and underway. NTC, non-template control; WT, wild type.

Supplementary Figure 4 Timing of events during the first cell cycle of a fertilized mouse embryo.

Diagram illustrating the timing of critical events during the first cell cycle of a fertilized mouse embryo, including morphological changes, approximate timing of cell cycle stage and molecular hallmarks that lead up to the first cleavage event. As the exact timing of insemination is not known, a reasonable suggestion would be to regard hour 0 as midnight. The first zygotic cell cycle culminates approximately 21 hours post-fertilization, allowing a short window as the ideal timing for CRISPR-EZ editing. NHEJ is the predominate repair mechanism active during G1, S and G2 phase of cell cycle while HDR repair demonstrates peak activity in late S and G2 phases, when sister chromatin is most accessible. Thus, the ideal timing to deliver CRISPR editing machinery for both NHEJ and HDR is before entry into the first zygotic S-phase, prior to DNA replication, which occurs approximately 10-11 hours post-fertilization. This would roughly translate as conducting CRISPR-EZ between 7-11 AM. In light of these timelines, pre-assembled Cas9/sgRNA RNPs have several advantages over cas9 mRNA/sgRNA, as the RNPs catalyze rapid editing on a diploid pronucleus genome, while the absence of sustained Cas9 translation minimizes potential off-target effects. With our optimized CRISPR-EZ protocol, the majority of pronuclear embryos have just begun to enter S-Phase when the Cas9 RNPs are delivered by electroporation. Consistent with Kim et al 2014, our experimentally-derived kinetics within the embryo suggest that the first signs of editing occur 2-4 hours post-electroporation, around the time when HDR is anticipated to be active. Thus, the CRISPR-EZ protocol is optimized towards facilitating both NHEJ and HDR outcomes.

1. Heyer, W.-D., Ehmsen, K. T. & Liu, J. Regulation of homologous recombination in eukaryotes. Annu. Rev. Genet. 44, 113–39 (2010).

2. Kim, S., Kim, D., Cho, S. W., Kim, J. J.-S. J.-S. & Kim, J. J.-S. J.-S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

Supplementary Figure 5 Preparing a mouth pipette.

(a) Glass transfer pipets are made by carefully bringing the center of a glass capillary tube into a medium height flame. (b) As the glass capillary tube begins to melt, quickly lift the tube out of the flame and then pull the tube outward about 2-3 inches. (c) The pulled tube is then snapped in the middle, producing two glass transfer pipets. (d) The glass transfer pipet is then inserted into the clear pipet holder of the mouth pipet.

Supplementary Figure 6 Diagram illustrating how to set up an embryo culture dish.

(a) Top and side views of a 35 mm plate used for embryo culture are shown, 20-30 μl of KSOM+BSA droplets are evenly distributed with a handheld pipet. These droplets are then carefully overlaid with mineral oil delivered by serological pipet. Droplets will maintain their integrity due to different viscosity than the overlaid mineral oil. The plate is then allowed to equilibrate for at least 4 hours (preferably overnight) in a cell culture incubator. Embryos are then placed into droplets by piercing the mineral oil with a glass mouth pipet so as to minimally perturb the equilibrated culturing environment. (b) Photographic images of embryo culture plates are shown.

Supplementary Figure 7 Diagram illustrating how to set up an embryo wash dish.

Top and side views of a 60 mm plate used for embryo washes are shown, 50 μl of M2+BSA droplets are distributed with a handheld pipet. The pattern shown is to maximize the number of wash droplets per plate. The plate can be left in 37°C incubator conditions prior to use so that embryos are not exposed to non-physiological temperatures for very long.

Supplementary Figure 8 Potential issues during sgRNA quality control.

Bioanalyzer traces illustrate typical experimental issues observed during sgRNA synthesis and quality control. (a) Loading marker; (b) Primer dimers or severe sgRNA degradation; (c) Multiple sgRNA synthesis products, possibly due to mis-priming; (d) An example of sgRNA that exhibits a slight shoulder forming from the main peak. While this profile is slightly deviated from that of a perfect sgRNA synthesis, their editing efficiency is comparable (e) Insufficient heat denaturation of good quality sgRNAs prior to loading; (f) A representative profile for an ideal sgRNA synthesis.

Supplementary Figure 9 Potential issues during embryo collection and culture.

Representative images of newly ovulated oocytes and embryos. Viable embryos display a distinct oolema boundary that is easily distinguished from the zona pellucida (green arrows). Inviable embryos take on an amorphous appearance where it is difficult to identify a specific cellular boundary (red arrows). Even at the highest magnification shown (40x), it is difficult to discern oocytes from fertilized pronucleus stage embryos by the presence of male and female pronucleus. Scale Bars: 50 μm.

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Modzelewski, A., Chen, S., Willis, B. et al. Efficient mouse genome engineering by CRISPR-EZ technology. Nat Protoc 13, 1253–1274 (2018). https://doi.org/10.1038/nprot.2018.012

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