Abstract
The discovery of sequence specific nucleases such as ZFNs, TALENs, and CRISPR/Cas9 has revolutionized genome editing. The CRISPR/Cas9 system has particularly emerged as a highly simple and efficient approach towards generating genome-edited animal models of most of the experimental species. The limitation of these novel genome editing tools is that, till date, they depend on traditional pronuclear injection (PI)-based transgenic technologies developed over the last three decades. PI requires expensive micromanipulator systems and the equipment operators must possess a high level of skill. Therefore, since the establishment of PI-based transgenesis, various research groups worldwide have attempted to develop alternative and simple gene delivery methods. However, owing to the failure of chromosomal integration of the transgene, none of these methods gained the level of confidence as that by the PI method in order to be adapted as a routine approach. The recently developed genome editing systems do not require complicated techniques. Therefore, presently, attention is being focused on non-PI-based gene delivery into germ cells for simple and rapid production of genetically engineered animals. For example, a few reports during the previous 1–2 years demonstrated the use of electroporation (EP) in isolated zygotes that helped to overcome the absolute dependency on PI techniques. Recently, another breakthrough technology called genome editing via oviductal nucleic acids delivery (GONAD) that directly delivers nucleic acids into zygotes within the oviducts in situ was developed. This technology completely relieves the bottlenecks of animal transgenesis as it does not require PI and ex vivo handling of embryos. This review discusses in detail the in vivo gene delivery methods targeted towards female reproductive tissues as these methods that have been developed over the past 2–3 decades can now be re-evaluated for their suitability to deliver the CRISPR/Cas9 components to produce transgenic animals. This review also provides an overview of the latest advances in CRISPR-enabled delivery technologies that have caused paradigm shifts in animal transgenesis methodologies.
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References
Aida T, Chiyo K, Usami T, Ishikubo H, Imahashi R, Wada Y, Tanaka KF, Sakuma T, Yamamoto T, Tanaka K (2015) Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biol 16:87
Bedrosian JC, Gratton MA, Brigande JV, Tang W, Landau J, Bennett J (2006) In vivo delivery of recombinant viruses to the fetal murine cochlea: transduction characteristics and long-term effects on auditory function. Mol Ther 14:328–335
Besenfelder U, Havlicek V, Mösslacher G, Brem G (2001) Collection of tubal stage bovine embryos by means of endoscopy. A technique report. Theriogenology 55:837–845
Besenfelder U, Havlicek V, Moesslacher G, Gilles M, Tesfaye D, Griese J, Hoelker M, Hyttel PM, Laurincik J, Brem G, Schellander K (2008) Endoscopic recovery of early preimplantation bovine embryos: effect of hormonal stimulation, embryo kinetics and repeated collection. Reprod Dom Anim 43:566–572
Besenfelder U, Havlicek V, Kuzmany A, Brem G (2010) Endoscopic approaches to manage in vitro and in vivo embryo development: use of the bovine oviduct. Theriogenology 73:768–776
Carlson DF, Tan W, Lillico SG, Stverakova D, Proudfoot C, Christian M, Voytas DF, Long CR, Whitelaw CB, Fahrenkrug SC (2012) Efficient TALEN-mediated gene knockout in livestock. Proc Natl Acad Sci USA 109:17382–17387
Chen XG, Zhu HZ, Gong JL, Li F, Xue JL (2004) Efficient delivery of human clotting factor IX after injection of lentiviral vectors in utero. Acta Pharmacol Sin 25:789–793
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Cornford EM, Hyman S, Cornford ME, Chytrova G, Rhee J, Suzuki T, Yamagata T, Yamakawa K, Penichet ML, Pardridge WM (2015) Non-invasive gene targeting to the fetal brain after intravenous administration and transplacental transfer of plasmid DNA using PEGylated immunoliposomes. J Drug Target 2:1–10
Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM (1996) Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383:531–535
Efremov AM, Buglaeva AO, Orlov SV, Burov SV, Ignatovich IA, Dizhe EB, Shavva VS, Perevozchikov AP (2010) Transfer of genetic constructions through the transplacental barrier into mice embryos. Russ J Dev Biol 41:71–76
Endo M, Zoltick PW, Chung DC, Bennett J, Radu A, Muvarak N, Flake AW (2007) Gene transfer to ocular stem cells by early gestational intraamniotic injection of lentiviral vector. Mol Ther 15:579–587
Fujii W, Kawasaki K, Sugiura K, Naito K (2013) Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucl Acids Res 41:e187
Gaensler KM, Tu G, Bruch S, Liggitt D, Lipshutz GS, Metkus A, Harrison M, Heath TD, Debs RJ (1999) Fetal gene transfer by transuterine injection of cationic liposome-DNA complexes. Nat Biotechnol 17:1188–1192
Garcia-Frigola C, Carreres MI, Vegar C, Herrera E (2007) Gene delivery into mouse retinal ganglion cells by in utero electroporation. BMC Dev Biol 7:103
Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM, Jenkins SS, Wood A, Cui X, Meng X, Vincent A, Lam S, Michalkiewicz M, Schilling R, Foeckler J, Kalloway S, Weiler H, Ménoret S, Anegon I, Davis GD, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Jacob HJ, Buelow R (2009) Knockout rats via embryo microinjection of zinc finger nucleases. Science 24:325–433
Gordon JW (2001) Direct exposure of mouse ovaries and oocytes to high doses of an adenovirus gene therapy vector fails to lead to germ cell transduction. Mol Ther 3:557–564
Gubbels SP, Woessner DW, Mitchell JC, Ricci AJ, Brigande JV (2008) Functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer. Nature 455:537–541
Gurumurthy CB, Takahashi G, Wada K, Miura H, Sato M, Ohtsuka M (2016) GONAD: a novel CRISPR/Cas9 genome editing method that does not require ex vivo handling of embryos. Curr Protoc Hum Genet 88:15.8.1–15.8.12
Hai T, Teng F, Guo R, Li W, Zhou Q (2014) One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res 24:372–375
Harrison MM, Jenkins BV, O’Connor-Giles KM, Wildonger JA (2014) CRISPR view of development. Genes Dev 28:1859–1872
Hashimoto M, Takemoto T (2015) Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Sci Rep 5:11315
Henriques-Coelho T, Gonzaga S, Endo M, Zoltick PW, Davey M, Leite-Moreira AF, Correia-Pinto J, Flake AW (2007) Targeted gene transfer to fetal rat lung interstitium by ultrasound-guided intrapulmonary injection. Mol Ther 15:340–347
Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278
Kaneko T, Mashimo T (2015) Simple genome editing of rodent intact embryos by electroporation. PLoS ONE 10:e0142755
Kaneko T, Sakuma T, Yamamoto T, Mashimo T (2014) Simple knockout by electroporation of engineered endonucleases into intact rat embryos. Sci Rep 4:6382
Kikuchi N, Nakamura S, Ohtsuka M, Kimura M, Sato M (2002) Possible mechanism of gene transfer into early to mid-gestational mouse fetuses by tail vein injection. Gene Ther 9:1529–1541
Kobayashi T, Namba M, Koyano T, Fukushima M, Sato M, Ohtsuka M, Matsuyama M (2018) Successful production of genome-edited rats by the rGONAD method. BMC Biotechnol 18:19
Koyama S, Kimura T, Ogita K, Nakamura H, Tabata C, Md Abu Hadi Noor Ali K, Temma-Asano K, Shimoya K, Tsutsui T, Koyama M, Kaneda Y, Murata Y (2006) Simple and highly efficient method for transient in vivo gene transfer to mid-late pregnant mouse uterus. J Reprod Immunol 70:59–69
Laurema A, Heikkila A, Keski-Nisula L, Heikura T, Letholainen P, Manninen H, Tuomisto TT, Heinonen S, Yla-Herttuala S (2003) Transfection of oocytes and other types of ovarian cells in rabbits after a direct injection into uterine arteries of adenoviruses and plasmid/liposomes. Gene Ther 10:580–584
Li W, Teng F, Li T, Zhou Q (2013) Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR–Cas systems. Nat Biotechnol 31:684–686
Ma Y, Ma J, Zhang X, Chen W, Yu L, Lu Y, Bai L, Shen B, Huang X, Zhang L (2014) Generation of eGFP and Cre knockin rats by CRISPR/Cas9. FEBS J 281:3779–3790
Mali P, Yang L, Esvelt KM, Aach J (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826
Meyera M, Hrabé de Angelisb M, Wursta W, Kühna R (2010) Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. Proc Natl Acad Sci USA 107:15022–15026
Nakahira E, Yuasa S (2005) Neuronal generation, migration, and differentiation in the mouse hippocampal primoridium as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation. J Comp Neurol 483:329–340
Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J (2014) Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156:836–843
Ohtsuka M, Sato M, Miura H, Takabayashi S, Matsuyama M, Koyano T, Arifin N, Nakamura S, Wada K, Gurumurthy CB (2018) i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases. Genome Biol 19:25
Okuda K, Xin KQ, Haruki A, Kawamoto S, Kojima Y, Hirahara F, Okada H, Klinman D, Hamajima K (2001) Transplacental genetic immunization after intravenous delivery of plasmid DNA to pregnant mice. J Immunol 167:5478–5484
O’shea KS, De Boer LS, Slawny NA, Gratsch TE (2006) Transplacental RNAi: deciphering gene function in the postimplantation-staged embryo. J Biomed Biotechnol 4:18657
Qin W, Dion SL, Kutny PM, Zhang Y, Cheng AW, Jillette NL, Malhotra A, Geurts AM, Chen Y-G, Wang H (2015) Efficient CRISPR/Cas9-mediated genome editing in mice by zygote electroporation of nuclease. Genetics 200:423–430
Rahim AA, Wong AMS, Howe SJ, Buckley SMK, Acosta-Saltos AD, Elston KE, Ward NJ, Philpott NJ, Cooper JD, Anderson PN, Waddington SN, Thrasher AJ, Raivich G (2009) Efficient gene delivery to the adult and fetal CNS using pseudotyped non-integrating lentiviral vectors. Gene Ther 16:509–520
Relloso M, Esponda P (1998) In vivo gene transfer to the mouse oviduct epithelium. Fertil Steril 70:366–368
Relloso M, Esponda P (2000) In-vivo transfection of the female reproductive tract epithelium. Mol Hum Reprod 6:1099–1105
Rios M, Venegas A, Croxatto HB (2002) In vivo expression of beta-galactosidase by rat oviduct exposed to naked DNA or messenger RNA. Biol Res 35:333–338
Ru R, Yao Y, Yu S, Yin B, Xu W, Zhao S, Qin L, Chen X (2013) Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs. Cell Regen 2:5
Saito T (2006) In vivo electroporation in the embryonic mouse central nervous system. Nat Protoc 1:1552–1558
Saito T, Nakatsuji N (2001) Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol 240:237–246
Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T (2014) Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sci Rep 4:5400
Sakurai T, Watanabe S, Kamiyoshi A, Sato M, Shindo T (2014) A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice. BMC Biotechnol 14:69
Sander JD, Cade L, Khayter C, Reyon D, Peterson RT, Joung JK, Yeh JR (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat Biotechnol 29:697–698
Sato M (2005) Intraoviductal introduction of plasmid DNA and subsequent electroporation for efficient in vivo gene transfer to murine oviductal epithelium. Mol Reprod Dev 71:321–330
Sato M, Tanigawa M, Kikuchi N, Nakamura S, Kimura M (2003) Efficient gene delivery into murine ovarian cells by intraovarian injection of plasmid DNA and subsequent in vivo electroporation. Genesis 35:169–174
Sato M, Tanigawa M, Kikuchi N (2004) Non-viral gene transfer to surface skin of mid-gestational murine embryos by intraamniotic injection and subsequent electroporation. Mol Reprod Dev 69:268–277
Sato M, Akasaka E, Saitoh I, Ohtsuka M, Watanabe S (2012) In vivo gene transfer in mouse preimplantation embryos after intraoviductal injection of plasmid DNA and subsequent in vivo electroporation. Syst Biol Reprod Med 58:278–287
Sato M, Ohtsuka M, Watanabe W, Gurumurthy CB (2016) Nucleic acids delivery methods for genome editing in zygotes and embryos: the old, the new, and the old–new. Biol Direct 11:16
Shen B, Zhang J, Wu H, Wang J, Ma K, Li Z, Zhang X, Zhang P, Huang X (2013) Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res 23:720–723
Shimizu T, Miyahayashi Y, Yokoo M, Hoshino Y, Sasada H, Sato E (2004) Molecular cloning of porcine growth differentiation factor 9 (GDF-9) cDNA and its role in early folliculogenesis: direct ovarian injection of GDF-9 gene fragments promotes early folliculogenesis. Reproduction 128:537–543
Shinmyo Y, Tanaka S, Tsunoda S, Hosomichi K, Tajima A, Kawasaki H (2016) CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation. Sci Rep 6:20611
Soma M, Aizawa H, Ito Y, Maekawa M, Osumi N, Nakahira E, Okamoto H, Tanaka K, Yuasa S (2009) Development of the mouse amygdala as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation. J Comp Neurol 513:113–128
Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, Hatanaka F, Yamamoto M, Araoka T, Li Z, Kurita M, Hishida T, Li M, Aizawa E, Guo S, Chen S, Goebl A, Soligalla RD, Qu J, Jiang T, Fu X, Jafari M, Esteban CR, Berggren WT, Lajara J, Nuñez-Delicado E, Guillen P, Campistol JM, Matsuzaki F, Liu GH, Magistretti P, Zhang K, Callaway EM, Zhang K, Belmonte JC (2016) In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540:144–149
Tabata H, Nakajima K (2001) Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex. Neuroscience 103:865–872
Takahashi M, Nomura T, Osumi N (2008) Transferring genes into cultured mammalian embryos by electroporation. Dev Growth Differ 50:485–497
Takahashi G, Gurumurthy CB, Wada K, Miura H, Sato M, Ohtsuka M (2015) GONAD: genome-editing via Oviductal Nucleic Acids Delivery system: a novel microinjection independent genome engineering method in mice. Sci Rep 5:11406
Tsukamoto M, Ochiya T, Yoshida S, Sugimura T, Terada M (1995) Gene transfer and expression in progeny after intravenous DNA injection into pregnant mice. Nat Genet 9:243–248
Tsunekawa Y, Terhune RK, Fujita I, Shitamukai A, Suetsugu T, Matsuzaki F (2016) Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain. Development 143:3216–3222
Velazquez MA, Kues WA (2011) In vivo gene transfer in the female bovine: potential applications for biomedical research in reproductive sciences. In: Komorowska MA, Olsztynska-Janus S (eds) Biomedical Engineering, Trends, Research and Technologies. Rijeka, HR. InTech, pp 217–244
Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918
Whitworth KM, Lee K, Benne JA, Beaton BP, Spate LD, Murphy SL, Samuel MS, Mao J, O’Gorman C, Walters EM, Murphy CN, Driver J, Mileham A, McLaren D, Wells KD, Prather RS (2014) Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. Biol Reprod 91:78
Woo YJ, Raju GP, Swain JL, Richmond ME, Gardner TJ, Balice-Gordon RJ (1997) In utero cardiac gene transfer via intraplacental delivery of recombinant adenovirus. Circulation 96:3561–3569
Yang S-Y, Wang J-G, Cui H-X, Sun S-G, Li Q, Gu L, Hong Y, Liu P-P, Liu W-Q (2007) Efficient generation of transgenic mice by direct intraovarian injection of plasmid DNA. Biochem Biophys Res Commun 358:266–271
Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154:1370–1379
Zanjani ED, Anderson WF (1999) Prospects for in utero human gene therapy. Science 285:2084–2088
Zhu X, Xu Y, Yu S, Lu L, Ding M, Cheng J, Song G, Gao X, Yao L, Fan D, Meng S, Zhang X, Hu S, Tian Y (2014) An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system. Sci Rep 4:6420
Acknowledgements
This study was partially supported by a Grant (No. 24580411 for Masahiro Sato; No. 15K14371 for Masato Ohtsuka; No. 16H05049 for Shingo Nakamura) from the Ministry of Education, Science, Sports, and Culture, Japan.
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Sato, M., Ohtsuka, M., Nakamura, S. et al. In vivo genome editing targeted towards the female reproductive system. Arch. Pharm. Res. 41, 898–910 (2018). https://doi.org/10.1007/s12272-018-1053-z
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DOI: https://doi.org/10.1007/s12272-018-1053-z