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Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD

Nature Protocols volume 14, pages 2452–2482 (2019)Cite this article

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Abstract

Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps. GONAD involves injection of CRISPR components (Cas9 mRNA and guide RNA (gRNA)) into the oviducts of pregnant females 1.5 d post conception, followed by in vivo electroporation to deliver the components into the zygotes in situ. Using GONAD, we demonstrated that target genes can be disrupted and analyzed at different stages of mouse embryonic development. Subsequently, we developed improved GONAD (i-GONAD) by delivering CRISPR ribonucleoproteins (RNPs; Cas9 protein or Cpf1 protein and gRNA) into day-0.7 pregnant mice, which made it suitable for routine generation of knockout and large-deletion mouse models. i-GONAD can also generate knock-in models containing up to 1-kb inserts when single-stranded DNA (ssDNA) repair templates are supplied. i-GONAD offers other advantages: it does not require vasectomized males and pseudo-pregnant females, the females used for i-GONAD are not sacrificed and can be used for other experiments, it can be easily adopted in laboratories lacking sophisticated microinjection equipment, and can be implemented by researchers skilled in small-animal surgery but lacking embryo-handling skills. Here, we provide a step-by-step protocol for establishing the i-GONAD method. The protocol takes ∼6 weeks to generate the founder mice.

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Fig. 1: Overview of the i-GONAD workflow.
Fig. 2: Equipment and instruments required for i-GONAD procedure.
Fig. 3: Examples of 1% (wt/vol) agarose gels showing electrophoresis.
Fig. 4: Diagrams showing the surgical steps to expose ovary/oviduct/uterus (Steps 10–16).
Fig. 5: Diagrams showing loading of genome-editing reagents into an injection needle (Steps 17 and 18).
Fig. 6: Diagrams showing intraoviductal injection (Step 20).
Fig. 7: Diagrams showing in vivo electroporation (Steps 22–26).
Fig. 8: Diagrams showing steps after in vivo electroporation (Steps 27–30).

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Data availability

All the data generated and analyzed in this study are included in the tables, figures and supplementary material.

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Acknowledgements

We thank Y. Ishikawa (Tokai University) for preparing pregnant MCH(ICR) mice and BEX Co. Ltd for advising us about electroporation optimization. This work was supported by the 2014 Tokai University School of Medicine Research Aid, the Research and Study Project of Tokai University General Research Organization, the 2016–2017 Tokai University School of Medicine Project Research, the Research Aid from the Institute of Medical Sciences inTokai University, the MEXT‐Supported Program for the Strategic Research Foundation at Private Universities 2015–2019, and a Grant-in-Aid for Challenging Exploratory Research (15K14371) from JSPS to M.O. AsCpf1 protein was a gift fromIDT. We thank J. M. Miano (University of Rochester) and G. Burgio (Australian National University) for their helpful comments on the manuscript.

Author information

Author notes

  1. These authors contributed equally: Channabasavaiah B. Gurumurthy, Masahiro Sato, Masato Ohtsuka.

Authors and Affiliations

  1. Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA

    Channabasavaiah B. Gurumurthy

  2. Department of Pharmacology and Experimental Neuroscience, College of Medicine, University of Nebraska Medical Center, Omaha, NE, USA

    Channabasavaiah B. Gurumurthy

  3. Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan

    Masahiro Sato

  4. Support Center for Medical Research and Education, Tokai University, Kanagawa, Japan

    Ayaka Nakamura & Sanae Ogiwara

  5. Laboratory of Animal Regeneration Systemology, Department of Life Science, School of Agriculture, Meiji University, Kanagawa, Japan

    Masafumi Inui

  6. Meiji University International Institute for Bio-Resource Research, Kanagawa, Japan

    Masafumi Inui

  7. Laboratory of Regulatory Biology, Department of Life Science, School of Agriculture, Meiji University, Kanagawa, Japan

    Natsuko Kawano

  8. Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa, Japan

    Md Atiqul Islam, Hiromi Miura & Masato Ohtsuka

  9. Laboratory of Laboratory Animal Science and Medicine, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan

    Md Atiqul Islam

  10. Laboratory for Animal Resources Development, Hamamatsu University School Of Medicine, Hamamatsu, Shizuoka, Japan

    Shuji Takabayashi

  11. Division of Molecular Genetics, Shigei Medical Research Institute, Minami-ku, Okayama, Japan

    Makoto Matsuyama

  12. RNA Biology Laboratory, Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan

    Shinichi Nakagawa

  13. Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Kanagawa, Japan

    Hiromi Miura & Masato Ohtsuka

  14. The Institute of Medical Sciences, Tokai University, Kanagawa, Japan

    Masato Ohtsuka

Authors
  1. Channabasavaiah B. Gurumurthy
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Contributions

M.O., C.B.G. and M.S. conceived the idea of i-GONAD. M.O., M.S. and M.A.I. were involved in testing the 2-d protocol. H.M. established the long ssDNA preparation system. M.I. and N.K. tested the hand-made capillary-based i-GONAD method. M.O., A.N. and S.O. optimized i-GONAD in the C57BL/6 strain. S.N. tested the squeezing steps in the i-GONAD procedure. M.O., S.T. and M.M. performed and optimized i-GONAD in several mouse strains and electroporators. C.B.G., M.S., M.I., M.A.I., S.N., H.M. and M.O. contributed to the writing of the manuscript.

Corresponding authors

Correspondence to Channabasavaiah B. Gurumurthy or Masato Ohtsuka.

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Competing interests

C.B.G., M.S. and M.O. have filed a patent application relating to the work described in this paper with International Patent Application no. PCT/US2018/047748, filed August 23, 2017 (Methods and compositions for in situ germline genome engineering). Tokai University and BEX Co. Ltd. applied for a patent describing the electroporation condition using CUY21Edit II on application number 2017–233100 (filed December 5, 2017). M.O. is an inventor of the patent.

Additional information

Peer review information: Nature Protocols thanks Izuho Hatada, Michael Wiles and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Ohtsuka, M. et al. Genome Biol. 19, 25 (2018): https://doi.org/10.1186/s13059-018-1400-x

Takahashi, G. et al. Sci. Rep. 5, 11406 (2015): https://doi.org/10.1038/srep11406

Miura, H., Quadros, R. M., Gurumurthy, C. B. & Ohtsuka, M. Nat. Protoc. 13, 195–215 (2018): https://doi.org/10.1038/nprot.2017.153

Integrated supplementary information

Supplementary Fig. 1 Preparation of devises used for intraoviductal injection.

(a) Cartoon showing various parts of a mouth pipetting devise. (b) Cartoon showing glass capillaries needed for intraoviductal injection. Drawings of a capillary tip before and after it was cut with the microscissors.

Supplementary Fig. 2 Dissecting microscopic view of oviduct after injection of solution containing Fast Green FCF.

Images of the oviduct photographed immediately after injection (a), and after covering it with a wet Kimwipe towel (b).

Supplementary Fig. 3 Genome-editing efficiency and number of fetuses retrieved after performing i-GONAD with different electroporation currents.

Tyr rescue experiment is shown as an example. Two MCH(ICR) females were used for each current value. Gray bar indicates a total number of mid-gestation fetuses recovered from two females. Yellow bar indicates a total number of genome modified fetuses (includes both knock-in and indel). Blue bar indicates a total number of fetuses with pigmentation (successful knock-in). Green line indicates the % of genome edited fetuses. The data of this experiment are available from the corresponding author upon request.

Supplementary Fig. 4 Injection of solution using hand-made glass capillary.

(a) Dissecting microscopic view of injection (see also Supplementary Video 3). (b) Tip of hand-made glass capillary.

Supplementary Fig. 5 Cartoons showing collection of 2-cell-stage embryos from the i-GONAD-treated mouse (procedure 1 of 2).

(a) Making a midline incision at the ventral surface of the euthanized mouse. (b) Opening the ventral skin. (c) Opening the ventral muscle. (d) Pulling out of both ovaries/oviducts/uteri and trimming off the portion of the uterus. (e) Placing of the dissected ovaries/oviducts/uteri and adding a small amount of 1x PBS onto the tissues. (f) Removing of the associated blood on ovary/oviduct/uterus by placing them onto a paper towel. (g) Removing the mesenterium (a membranous fold) associated with the uterus and oviduct. (h) Removing the adipose tissue associated with an ovary. (i) Removing the ovary using microscissors under a dissecting microscope. R, right; L, left.

Supplementary Fig. 6 Cartoons showing collection of 2-cell-stage embryos from the i-GONAD-treated mouse (procedure 2 of 2).

(a) and (b) Cutting of the junctional portion between oviduct and uterus using microscissors. (c) Inserting the 30-G needle attached with a 1-ml disposable plastic syringe into the oviductal lumen. (d) Flushing the contents while watching the flowing out of embryos. (e) Removing the oviduct and collecting the released embryos using an egg handling pipette. The picture above the drops (top right) is the side view of the embryo handling procedure in the drop. Washing of the collected embryos by passing them through small drops, and (f) transferring them into another fresh drop ready for observing under an inverted fluorescence microscope. Embryos obtained from four females (#1 to #4) are shown as an example. R, embryos from right oviduct; L, embryos from left oviduct. Right most image shows a side view of microscopic observation.

Supplementary Fig. 7 Fluorescence in the preimplantation mouse embryos after i-GONAD.

(a) - (c) Two-cell embryos collected from the MCH(ICR) females that were subjected to i-GONAD procedure following injection of a solution containing tetramethylrhodamine-labeled dextran 3kDa (red), EGFP mRNA (green). Note that the embryos are observed for both red and green fluorescence. (d) to (f) Two-cell embryos collected from the MCH(ICR) females similarly treated except the electroporation step showing no fluorescence in any of the embryos. Scale bar = 100 µM.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7

Reporting Summary

Supplementary Video 1

Surgical exposure of an oviduct.

Supplementary Video 2

Loading solution into a capillary needle.

Supplementary Video 3

Injecting solution into an oviduct by using a hand-made capillary with injection choice 1 (Fig. 6).

Supplementary Video 4

Injecting solution into an oviduct and then performing electroporation on the oviduct with an NEPA21 electroporator and CUY652-3 electrodes (2-d protocol) using injection choice 1 (Fig. 6).

Supplementary Video 5

Squeezing of the ampulla to disperse the injected solution uniformly within the lumen.

Supplementary Video 6

Injecting solution into an oviduct and then performing electroporation on the oviduct with a CUY21EditII electroporator and LF650P3 electrodes using injection choice 1 (Fig. 6).

Supplementary Video 7

Preparing a glass micropipette with a flame.

Supplementary Video 8

Cutting the tip of a glass capillary.

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Gurumurthy, C.B., Sato, M., Nakamura, A. et al. Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD. Nat Protoc 14, 2452–2482 (2019). https://doi.org/10.1038/s41596-019-0187-x

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