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CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos

Nature Protocols volume 9, pages 2493–2512 (2014)Cite this article

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Abstract

Conventional embryonic stem cell (ESC)–based gene targeting, zinc-finger nuclease (ZFN) and transcription activator–like effector nuclease (TALEN) technologies are powerful strategies for the generation of genetically modified animals. Recently, the CRISPR/Cas system has emerged as an efficient and convenient alternative to these approaches. We have used the CRISPR/Cas system to generate rat strains that carry mutations in multiple genes through direct injection of RNAs into one-cell embryos, demonstrating the high efficiency of Cas9-mediated gene editing in rats for simultaneous generation of compound gene mutant models. Here we describe a stepwise procedure for the generation of knockout and knock-in rats. This protocol provides guidelines for the selection of genomic targets, synthesis of guide RNAs, design and construction of homologous recombination (HR) template vectors, embryo microinjection, and detection of mutations and insertions in founders or their progeny. The procedure from target design to identification of founders can take as little as 6 weeks, of which <10 d is actual hands-on working time.

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Figure 1: Schematic of RNA-guided Cas9 nuclease.
Figure 2: DSB repair promotes gene editing.
Figure 3: Workflow for production of mutant rats using the CRISPR/Cas system.
Figure 4: PCR approach for sgRNA template production.
Figure 5: Schematic diagrams showing the strategy for constructing a rat gene-targeting vector.

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References

  1. Jacob, H.J., Lazar, J., Dwinell, M.R., Moreno, C. & Geurts, A.M. Gene targeting in the rat: advances and opportunities. Trends Genet. 26, 510–518 (2010).

    Article  CAS  Google Scholar 

  2. Thomas, K.R. & Capecchi, M.R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503–512 (1987).

    Article  CAS  Google Scholar 

  3. Koller, B.H., Marrack, P., Kappler, J.W. & Smithies, O. Normal development of mice deficient in beta 2M, MHC class I proteins, and CD8+ T cells. Science 248, 1227–1230 (1990).

    Article  CAS  Google Scholar 

  4. Schwartzberg, P.L., Goff, S.P. & Robertson, E.J. Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells. Science 246, 799–803 (1989).

    Article  CAS  Google Scholar 

  5. Mullins, L.J., Brooker, G. & Mullins, J.J. Transgenesis in the rat. Methods Mol. Biol. 180, 255–270 (2002).

    PubMed  Google Scholar 

  6. Lois, C., Hong, E.J., Pease, S., Brown, E.J. & Baltimore, D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–872 (2002).

    Article  CAS  Google Scholar 

  7. Zan, Y. et al. Production of knockout rats using ENU mutagenesis and a yeast-based screening assay. Nat. Biotechnol. 21, 645–651 (2003).

    Article  CAS  Google Scholar 

  8. Kitada, K. et al. Transposon-tagged mutagenesis in the rat. Nat. Methods 4, 131–133 (2007).

    Article  CAS  Google Scholar 

  9. Katter, K. et al. Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits. FASEB J. 27, 930–941 (2013).

    Article  CAS  Google Scholar 

  10. Tong, C., Li, P., Wu, N.L., Yan, Y. & Ying, Q.L. Production of p53 gene knockout rats by homologous recombination in embryonic stem cells. Nature 467, 211–213 (2010).

    Article  CAS  Google Scholar 

  11. Geurts, A.M. et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433 (2009).

    Article  CAS  Google Scholar 

  12. Cui, X. et al. Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat. Biotechnol. 29, 64–67 (2011).

    Article  CAS  Google Scholar 

  13. Tesson, L. et al. Knockout rats generated by embryo microinjection of TALENs. Nat. Biotechnol. 29, 695–696 (2011).

    Article  CAS  Google Scholar 

  14. Menoret, S. et al. Generation of Rag1-knockout immunodeficient rats and mice using engineered meganucleases. FASEB J. 27, 703–711 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Li, W., Teng, F., Li, T. & Zhou, Q. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nat. Biotechnol. 31, 684–686 (2013).

    Article  CAS  Google Scholar 

  17. Hu, X. et al. Heritable gene-targeting with gRNA/Cas9 in rats. Cell Res. 23, 1322–1325 (2013).

    Article  CAS  Google Scholar 

  18. Tong, C., Huang, G., Ashton, C., Li, P. & Ying, Q.L. Generating gene knockout rats by homologous recombination in embryonic stem cells. Nat. Protoc. 6, 827–844 (2011).

    Article  CAS  Google Scholar 

  19. Men, H. & Bryda, E.C. Derivation of a germline competent transgenic Fischer 344 embryonic stem cell line. PloS ONE 8, e56518 (2013).

    Article  CAS  Google Scholar 

  20. Men, H., Bauer, B.A. & Bryda, E.C. Germline transmission of a novel rat embryonic stem cell line derived from transgenic rats. Stem Cells Dev. 21, 2606–2612 (2012).

    Article  CAS  Google Scholar 

  21. Hirabayashi, M. et al. Establishment of rat embryonic stem cell lines that can participate in germline chimerae at high efficiency. Mol. Reprod. Dev. 77, 94 (2010).

    Article  CAS  Google Scholar 

  22. Hirabayashi, M. et al. Effect of leukemia inhibitory factor and forskolin on establishment of rat embryonic stem cell lines. J. Reprod. Dev. 60, 78–82 (2013).

    Article  Google Scholar 

  23. Hirabayashi, M. et al. Rat transgenesis via embryonic stem cells electroporated with the Kusabira-orange gene. Mol. Reprod. Dev. 77, 474 (2010).

    Article  CAS  Google Scholar 

  24. Bibikova, M., Beumer, K., Trautman, J.K. & Carroll, D. Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764 (2003).

    Article  CAS  Google Scholar 

  25. Miller, J.C. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25, 778–785 (2007).

    Article  CAS  Google Scholar 

  26. Kim, Y.G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. USA 93, 1156–1160 (1996).

    Article  CAS  Google Scholar 

  27. Mashimo, T. et al. Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID) using zinc-finger nucleases. PloS ONE 5, e8870 (2010).

    Article  Google Scholar 

  28. Porteus, M.H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003).

    Article  Google Scholar 

  29. Qiu, Z. et al. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases. Nucleic Acids Res. 41, e120 (2013).

    Article  CAS  Google Scholar 

  30. Wefers, B. et al. Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA. Nat. Protoc. 8, 2355–2379 (2013).

    Article  CAS  Google Scholar 

  31. Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 (2009).

    Article  CAS  Google Scholar 

  32. Moscou, M.J. & Bogdanove, A.J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).

    Article  CAS  Google Scholar 

  33. Sung, Y.H. et al. Knockout mice created by TALEN-mediated gene targeting. Nat. Biotechnol. 31, 23–24 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  36. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  38. Hwang, W.Y. et al. Heritable and precise zebrafish genome editing using a CRISPR-Cas system. PloS ONE 8, e68708 (2013).

    Article  CAS  Google Scholar 

  39. Cho, S.W., Kim, S., Kim, J.M. & Kim, J.S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).

    Article  CAS  Google Scholar 

  40. Shen, B. et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 23, 720–723 (2013).

    Article  CAS  Google Scholar 

  41. Garneau, J.E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010).

    Article  CAS  Google Scholar 

  42. Marraffini, L.A. & Sontheimer, E.J. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463, 568–571 (2010).

    Article  CAS  Google Scholar 

  43. Makarova, K.S. et al. Evolution and classification of the CRISPR-Cas systems. Nat. Rev. Microbiol. 9, 467–477 (2011).

    Article  CAS  Google Scholar 

  44. Mali, P., Esvelt, K.M. & Church, G.M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013).

    Article  CAS  Google Scholar 

  45. Sung, P. & Klein, H. Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat. Rev. Mol. Cell Biol. 7, 739–750 (2006).

    Article  CAS  Google Scholar 

  46. Lieber, M.R. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu. Rev. Biochem. 79, 181–211 (2010).

    Article  CAS  Google Scholar 

  47. Chen, F. et al. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat. Methods 8, 753–755 (2011).

    Article  CAS  Google Scholar 

  48. Richardson, C., Moynahan, M.E. & Jasin, M. Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations. Genes Dev. 12, 3831–3842 (1998).

    Article  CAS  Google Scholar 

  49. Kim, E. et al. Precision genome engineering with programmable DNA-nicking enzymes. Genome Res. 22, 1327–1333 (2012).

    Article  CAS  Google Scholar 

  50. Mashiko, D. et al. Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci. Rep. 3, 3355 (2013).

    Article  Google Scholar 

  51. Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013).

    Article  CAS  Google Scholar 

  52. Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  Google Scholar 

  53. 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).

    Article  CAS  Google Scholar 

  54. Fu, Y., Sander, J.D., Reyon, D., Cascio, V.M. & Joung, J.K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).

    Article  CAS  Google Scholar 

  55. Ran, F.A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    Article  CAS  Google Scholar 

  56. Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  Google Scholar 

  57. Xiao, A. et al. CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics 30, 1180–1182 (2014).

  58. Hwang, W.Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227–229 (2013).

    Article  CAS  Google Scholar 

  59. Deng, C. & Capecchi, M.R. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol. Cell. Biol. 12, 3365–3371 (1992).

    Article  CAS  Google Scholar 

  60. Brown, A.J. et al. Whole-rat conditional gene knockout via genome editing. Nat. Methods 10, 638–640 (2013).

    Article  CAS  Google Scholar 

  61. Ma, Y. et al. Generating rats with conditional alleles using CRISPR/Cas9. Cell Res. 24, 122–125 (2014).

    Article  CAS  Google Scholar 

  62. Bryksin, A.V. & Matsumura, I. Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids. BioTechniques 48, 463–465 (2010).

    Article  CAS  Google Scholar 

  63. Mark, A. & Suckow, S.H.W.a.C.L.F. The Laboratory Rat 2nd edn. (Academic Press, 2006).

  64. Brinster, R.L., Chen, H.Y., Trumbauer, M.E., Yagle, M.K. & Palmiter, R.D. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. USA 82, 4438–4442 (1985).

    Article  CAS  Google Scholar 

  65. Mangerich, A. et al. A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1. Trans. Res. 18, 261–279 (2009).

    Article  CAS  Google Scholar 

  66. Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R. Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, 2003).

  67. Si-Hoe, S.L., Wells, S. & Murphy, D. Production of transgenic rodents by the microinjection of cloned DNA into fertilized one-cell eggs. Mol. Biotechnol. 17, 151–182 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank F. Zhang of the Broad Institute of MIT and Harvard for kindly providing the Cas9 expression vector. We thank S. Siwko for comments and advice. This work was partially supported by grants from the State Key Development Programs of China (2010CB945403 to D.L. and 2012CB910400 to Mi.L.), grants from the National Natural Science Foundation of China (no. 31371455, 31171318 and 81330049) and a grant from the Science and Technology Commission of Shanghai Municipality (12XD1406100) and 14140900300.

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Author notes

  1. Yanjiao Shao, Yuting Guan and Liren Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China

    Yanjiao Shao, Yuting Guan, Liren Wang, Zhongwei Qiu, Meizhen Liu, Yuting Chen, Lijuan Wu, Yongmei Li, Xueyun Ma, Mingyao Liu & Dali Li

  2. Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas, USA

    Mingyao Liu

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Contributions

Y.S., Y.G., L. Wang, Z.Q., Me.L., Y.C., L. Wu and Y.L. performed the experiments; Y.S., Y.G., X.M., Mi.L. and D.L. analyzed the data; and Y.S., Y.G., Mi.L. and D.L. wrote the manuscript.

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Correspondence to Mingyao Liu or Dali Li.

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Integrated supplementary information

Supplementary Figure 1 In vitro validation of sgRNA activity.

4 nM PCR product containing the target sequence was mixed with purified H6MBP-Cas9-4NLS recombinant protein (2 nM final concentration) in NEB buffer 3 with (lane 2) or without (lane 1) corresponding gRNAs for 1 hour. After the incubation, the protein was discarded and the DNA was analyzed by agarose gel electrophoresis.

Supplementary Figure 2 Design of PCR primers for genotyping of knock-in mutations in rat genome.

(a) A primer pair (F and R) is designed outside of the homology arms of the ssODN. (b) Two primer pairs (5'F/5'R and 3'F/3'R) are designed. For each pair, one primer is located in the GOI (the inserted fragment). The other primer is located in rat genome outside the homology arms (left arm or right arm). About 400 bp extension of both left and right arm of the donor plasmid can be used as the positive control for testing the primers (optional).

Supplementary Figure 3 Generation of double mutant rats by co-injection of Cas9/sgRNAs targeting the Fah and Il2rg loci.

Detection of mutations in F0 rats generated by injection of Cas9/sgRNAs targeting Fah (a) and Il2rg (b) after T7EI digestion using PCR products amplified from RNA-injected F0 rat tail genomic DNA. Double mutant rats are indicated by arrowheads. M, DNA marker. (c) DNA Sequence of Fah and Il2rg loci double mutant rats. The target sites are in blue with PAM in red.

Supplementary Figure 4 Anticipated results for precise knock-in by ssODNs.

(a) A diagram of Cas9 nickase induced precise gene editing. The sequence of the target site and the ssODNs are listed. (b) Typical sequences of the founders after RNA/DNA injection. The PCR products were amplified with the primers outside of the homology region indicated in (a). Sequences were determined by DNA sequencing of individual TA clones of PCR products. The sgRNA target is underlined, the EcoRI site is in red and the LoxP site is in italics. (c) An example of a DNA sequencing result for precise HR.

Supplementary information

Supplementary Figure 1

In vitro validation of sgRNA activity. (PDF 142 kb)

Supplementary Figure 2

Design of PCR primers for genotyping of knock-in mutations in rat genome. (PDF 243 kb)

Supplementary Figure 3

Generation of double mutant rats by co-injection of Cas9/sgRNAs targeting the Fah and Il2rg loci. (PDF 238 kb)

Supplementary Figure 4

Anticipated results for precise knock-in by ssODNs. (PDF 244 kb)

Supplementary Method 1

Preparation of recombinant Cas9 protein. (PDF 163 kb)

Supplementary Method 2

Functional validation of sgRNA and Cas9 protein in vitro. (PDF 98 kb)

Supplementary Table 1

Summary of injection statistics in rat embryos from published reports. (PDF 177 kb)

Supplementary Table 2

Primer or oligo sequences. (PDF 174 kb)

Pronuclear microinjection

This video demonstrates the microinjection of the CRISPR-Cas system into the pronucleus of a rat zygote fixed with a holding pipette on the left. An obvious pronuclear swelling is shown to demonstrate the successful pronuclear injection. (MOV 2515 kb)

Supplementary Data 1: Full DNA sequence of pGS3-T7-sgRNA vector for in vitro synthesis of gRNA.

The pair of reverse located BbsI restriction sites used for cloning is indicated in bold and underlined. The DraI restriction site used to linearize the plasmid for run-off transcription is shown in bold, italicized text. The “stuffer” sequence that will eventually be replaced with the target-coding oligonucleotides is highlighted in yellow and the “scaffold” portion of sgRNA is shown as italicized underlined text. The sequence of an M13 primer binding site is shown as underlined text. (PDF 208 kb)

Supplementary Data 2: Full DNA sequence of SP6-Cas9 vector for in vitro mRNA synthesis.

The SP6 promoter sequence (highlighted in green) is followed by a Kozak sequence (highlighted in yellow) is subcloned into the Cas9-containing plasmid pX260 (Addgene plasmid# 42229) using NcoI restriction site (in red), NLS-hCas9-NLS-coding cassette is underlined. (PDF 185 kb)

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Shao, Y., Guan, Y., Wang, L. et al. CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos. Nat Protoc 9, 2493–2512 (2014). https://doi.org/10.1038/nprot.2014.171

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