Abstract
Engineering of the mouse germline is a key technology in biomedical research for studying the function of genes in health and disease. Since the first knockout mouse was described in 1989, gene targeting was based on recombination of vector encoded sequences in mouse embryonic stem cell lines and their introduction into preimplantation embryos to obtain germline chimeric mice. This approach has been replaced in 2013 by the application of the RNA-guided CRISPR/Cas9 nuclease system, which is introduced into zygotes and directly creates targeted modifications in the mouse genome. Upon the introduction of Cas9 nuclease and guide RNAs into one-cell embryos, sequence-specific double-strand breaks are created that are highly recombinogenic and processed by DNA repair enzymes. Gene editing commonly refers to the diversity of DSB repair products that include imprecise deletions or precise sequence modifications copied from repair template molecules. Since gene editing can now be easily applied directly in mouse zygotes, it has rapidly become the standard procedure for generating genetically engineered mice. This article covers the design of guide RNAs, knockout and knockin alleles, options for donor delivery, preparation of reagents, microinjection or electroporation of zygotes, and the genotyping of pups derived from gene editing projects.
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References
Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Mali P, Aach J, Stranges PB et al (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31:833–838
Wang H, Yang H, Shivalila CS et al (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918
Yang H, Wang H, Shivalila CS et al (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154:1370–1379
Kühn R (2021) Genome engineering in rodents – status quo and perspectives. Lab Anim 56:83–87
Tröder SE, Zevnik B (2021) History of genome editing: from meganucleases to CRISPR. Lab Anim 56:60–68
Qin W, Dion SL, Kutny PM et al (2015) Efficient CRISPR/Cas9-mediated genome editing in mice by zygote electroporation of nuclease. Genetics 200:423–430
Chen S, Lee B, Lee AY-F et al (2016) Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J Biol Chem 291:14457–14467
Lieber MR (2010) The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 79:181–211
Heyer W-D, Ehmsen KT, Liu J (2010) Regulation of homologous recombination in eukaryotes. Annu Rev Genet 44:113–139
Yao X, Zhang M, Wang X et al (2018) Tild-CRISPR allows for efficient and precise gene Knockin in mouse and human cells. Dev Cell 45:526–536
Yao X, Wang X, Hu X et al (2017) Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res 27:801–814
Quadros RM, Miura H, Harms DW et al (2017) 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
Codner GF, Mianné J, Caulder A et al (2018) Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants. BMC Biol 16:70
Lanza DG, Gaspero A, Lorenzo I et al (2018) Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles. BMC Biol 16:69
Yoon Y, Wang D, Tai PWL et al (2018) Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses. Nat Commun 9:412
Chen S, Sun S, Moonen D et al (2019) CRISPR-READI: efficient generation of knockin mice by CRISPR RNP electroporation and AAV donor infection. Cell Rep 27:3780–3789
Mizuno N, Mizutani E, Sato H et al (2018) Intra-embryo gene cassette Knockin by CRISPR/Cas9-mediated genome editing with adeno-associated viral vector. iScience 9:286–297
Lin X, Pelletier S, Gingras S et al (2016) CRISPR-Cas9-mediated modification of the NOD mouse genome with Ptpn22R619W mutation increases autoimmune diabetes. Diabetes 65:2134–2138
Tirado-Gonzalez I, Czlonka E, Nevmerzhitskaya A et al (2018) CRISPR/Cas9-edited NSG mice as PDX models of human leukemia to address the role of niche-derived SPARC. Leukemia 32:1049–1052
Boroviak K, Doe B, Banerjee R et al (2016) Chromosome engineering in zygotes with CRISPR/Cas9. Genesis 54:78–85
Birling M-C, Schaeffer L, André P et al (2017) Efficient and rapid generation of large genomic variants in rats and mice using CRISMERE. Sci Rep 7:43331
Fujii W, Kakuta S, Yoshioka S et al (2016) Zygote-mediated generation of genome-modified mice using Streptococcus thermophilus 1-derived CRISPR/Cas system. Biochem Biophys Res Commun 477:473–476
Kim Y, Cheong S-A, Lee JG et al (2016) Generation of knockout mice by Cpf1-mediated gene targeting. Nat Biotechnol 34:808–810
Zhang X, Liang P, Ding C et al (2016) Efficient production of gene-modified mice using Staphylococcus aureus Cas9. Sci Rep 6:32565
Hur JK, Kim K, Been KW et al (2016) Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat Biotechnol 34:807–808
Kim K, Ryu S-M, Kim S-T et al (2017) Highly efficient RNA-guided base editing in mouse embryos. Nat Biotechnol 35:435–437
Zuo E, Sun Y, Wei W et al (2019) Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364:289–292
Lee HK, Smith HE, Liu C et al (2020) Cytosine base editor 4 but not adenine base editor generates off-target mutations in mouse embryos. Commun Biol 3:19
Caso F, Davies B (2021) Base editing and prime editing in laboratory animals. Lab Anim 56:35–49
Liu Y, Li X, He S et al (2020) Efficient generation of mouse models with the prime editing system. Cell Discov 6:27
Shen MW, Arbab M, Hsu JY et al (2018) Predictable and precise template-free CRISPR editing of pathogenic variants. Nature 563:646–651
Allen F, Crepaldi L, Alsinet C et al (2018) Predicting the mutations generated by repair of Cas9-induced double-strand breaks. Nat Biotechnol 10:1038
Suzuki K, Tsunekawa Y, Hernandez-Benitez R et al (2016) In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540:144–149
Danner E, Lebedin M, de la Rosa K, Kühn R (2021) A homology independent sequence replacement strategy in human cells using a CRISPR nuclease. Open Biol 11:200283
Chu VT, Weber T, Graf R et al (2016) Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes. BMC Biotechnol 16:4
Low BE, Hosur V, Lesbirel S, Wiles MV (2022) Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase. Sci Reports 12:5424
Gurumurthy CB, O’Brien AR, Quadros RM et al (2019) Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation. Genome Biol 20:171
Haeussler M, Schönig K, Eckert H et al (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17:148
Dong Y, Li H, Zhao L et al (2019) Genome-wide off-target analysis in CRISPR-Cas9 modified mice and their offspring. G3 (Bethesda) 9:3645–3651
Iyer V, Shen B, Zhang W et al (2015) Off-target mutations are rare in Cas9-modified mice. Nat Methods 12:479
Anderson KR, Haeussler M, Watanabe C et al (2018) CRISPR off-target analysis in genetically engineered rats and mice. Nat Methods 15:512–514
Richardson CD, Kazane KR, Feng SJ et al (2018) CRISPR-Cas9 genome editing in human cells occurs via the Fanconi anemia pathway. Nat Genet 50:1132–1139
Richardson CD, Ray GJ, DeWitt MA et al (2016) Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol 34:339–344
Miura H, Quadros RM, Gurumurthy CB, Ohtsuka M (2018) Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat Protoc 13:195–215
Minev D, Guerra R, Kishi JY et al (2019) Rapid in vitro production of single-stranded DNA. Nucleic Acids Res 47:11956–11962
Hao M, Huang H, Hu Y, Qi H (2020) Construction of a system for single-stranded DNA isolation. Biotechnol Lett 42:1663–1671
Liang C, Li D, Zhang G et al (2015) Comparison of the methods for generating single-stranded DNA in SELEX. Analyst 140:3439–3444
Takeo T, Nakagata N (2015) Superovulation using the combined administration of inhibin antiserum and equine chorionic gonadotropin increases the number of ovulated oocytes in C57BL/6 female mice. PLoS One 10:e0128330
Beermann F, Hummler E, Schmid E, Schütz G (1993) Perinatal activation of a tyrosine aminotransferase fusion gene does not occur in albino lethal mice. Mech Dev 42:59–65
Brinkman EK, Kousholt AN, Harmsen T et al (2018) Easy quantification of template-directed CRISPR/Cas9 editing. Nucleic Acids Res 46:e58
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Wefers, B., Wurst, W., Kühn, R. (2023). Gene Editing in Mouse Zygotes Using the CRISPR/Cas9 System. In: Saunders, T.L. (eds) Transgenesis. Methods in Molecular Biology, vol 2631. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2990-1_8
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DOI: https://doi.org/10.1007/978-1-0716-2990-1_8
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