Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted primordial germ cells

Fenghua Zhang^{a, b},
Xianmei Li^{a, b},
Mudan He^{a, b},
Ding Ye^{a, b},
Feng Xiong^{a, b},
Golpour Amin^{a, b},
Zuoyan Zhu^{a, b},
Yonghua Sun^{a, b
, ,}

a.
State Key Laboratory of Freshwater Ecology and Biotechnology, Innovation Academy for Seed Design, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China
b.
College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China

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Corresponding author: E-mail address: yhsun@ihb.ac.cn (Yonghua Sun)

摘要

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Abstract: The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is efficient for generating different mutations, one of the main drawbacks is low survival rate during embryogenesis when knocking out some embryonic lethal genes. To overcome this problem, we developed a novel strategy using a combination of CRISPR/Cas9 mediated gene knockout with primordial germ cell (PGC) transplantation (PGCT) to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. Firstly, we optimized the procedure for CRISPR/Cas9 targeted PGCT by increasing the efficiencies of genome mutation in PGCs and induction of PGC fates in donor embryos for PGCT. Secondly, the optimized CRISPR/Cas9 targeted PGCT was utilized for generation of maternal-zygotic (MZ) mutants oftcf7l1a (gene essential for head development), pou5f3 (gene essential for zygotic genome activation) and chd (gene essential for dorsal development) at F₁ generation with relatively high efficiency. Finally, we revealed some novel phenotypes in MZ mutants of tcf7l1a and chd, as MZtcf7l1a showed elevated neural crest development while MZchd had much severer ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish. It is also feasible to speed up the genome editing in commercial fishes by utilizing a similar approach by surrogate production of CRISPR/Cas9 targeted germ cells.
- Zebrafish /
- CRISPR/Cas9 /
- Primordial germ cells /
- Transplantation /
- Maternal zygotic mutant

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Fig. 1. Poor survival of the embryos after Cas9/gRNA injection to knock out chd and pou5f3. A: Different phenotypes of the embryos after injection of 400 pg cas9-UTRsv40 or cas9-UTRnanos3 mRNA and 80 pg chd gRNA. C1: wild-type (WT) like; C2: ventralization. B: Statistics of embryos at corresponding phenotypes after knocking out chd by injection of cas9-UTRsv40 (n = 212) or cas9-UTRnanos3 mRNA and chd gRNA (n = 237). n, number of embryos calculated. C: Mutation rates of embryos at corresponding phenotypes after knocking out chd by injection of cas9-UTRsv40 or cas9-UTRnanos3 mRNA and chd gRNA. n, number of clones sequenced. D: Different phenotypes of the embryos after co-injection of 400 pg cas9-UTRsv40 or cas9-UTRnanos3 mRNA and 80 pg pou5f3 gRNA. C1: WT like; C2: dorsalization. E: Statistics of embryos at corresponding phenotypes after knocking out pou5f3 by injection of cas9-UTRsv40 (n = 251) or cas9-UTRnanos3 mRNA and pou5f3 gRNA (n = 248). n, number of embryos calculated. F: Mutation rates of embryos at corresponding phenotypes after knocking out pou5f3 by injection of cas9-UTRsv40 or cas9-UTRnanos3 mRNA and pou5f3 gRNA. n, number of clones sequenced. G: Statistics on survival rates of the embryos after injection of 400 pg cas9-UTRsv40 or cas9-UTRnanos3 mRNA and 80 pg gene specific gRNA to knock out chd and pou5f3 at 0, 2, 14, and 60 dpf.

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Fig. 2. Optimization of PGC-targeted mutagenesis and PGCT. A: Representative image of pre- and post-sorting of PGCs from a transgenic line of Tg(piwil1:egfp-UTRnanos3) at 2 dpf. B and C: Mutation efficiencies were calculated in somatic cells and PGCs after co-injection of cas9-UTRsv40 mRNA and gRNA (B) and cas9-UTRnanos3 mRNA and gRNA (C). Parallel experiments were done for three times. P < 0.01.D: Whole-mount in situ hybridization with vasa probe of the wild-type embryos and the embryos injected with 200 pg buc-UTRsv40 mRNA at 3 hpf. The embryos were animal pole view. Note that 8–10 vasa-positive cells appeared in the buc-injected embryos, compared with 4 vasa-positive cells in the control embryos. E: buc mRNA induced ectopic PGCs of donor embryos. Purple, orange and pink represent larva with less (PGC number≤25), moderate (25<PGC number≤35) and many (PGC number≥36) PGCs, respectively.F: Representative image showing a host embryo co-injected with 200 pg GFP-UTRnanos3 mRNA and 100 nM dnd morpholino ((MO), with complete loss of endogenous PGCs. G: Representative image showing a PGCs positive transplanted embryo screened at 35 hpf. The fluorescent PGCs from the donor embryos have been magnified at the bottom right corner.H: A image showing a PGCs positive transplanted embryo at 35 hpf, with mis-migrated PGCs (green arrow) and fluorescent somatic cells from donor embryo (white arrow). I: The success rate of PGCT, as indicated by PGC-positive transplanted embryos at 35 hpf, was significantly increased by injection of buc mRNA into the donor embryos. The experiment was replicated for three times. P < 0.01.J: The host embryos contained significantly higher number of PGCs compared with the control group, when using buc-overexpressed embryos as donors for PGCT. P < 0.001.

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Fig. 3. Efficient generation of MZ mutants of tcf7l1a by combination of CRISPR/Cas9 and PGCT. A: Schematic workflow represents process of the optimized procedure of PGC-targeted CRISPR/Cas9 and transplantation of induced PGCs. B: Mutation efficiencies of gametes of each mutated positive F₀ adult fish (4♀, 7♂). C: The phenotypes of offspring crossed by female #3 and male #7. C1 shows the WT like phenotype, C2 shows smaller eyes, C3 shows complete loss of eyes. D: tcf7l1a was barely expressed in mutants during early embryogenesis, compared with its high expression level in WT. E: The marker of telencephalon emx1 was not expressed in mutant embryos at early-somite stage; krox20, the marker for midbrain and hindbrain, was normally expressed in the mutants at early-somite stage; the expression of neural crest marker foxd3 was slightly increased in mutants at early-somite stage; the expression of six3b at telencephalon and eyes was strongly decreased in the mutant embryos at 24 hpf.

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Fig. 4. Efficient generation of MZ mutants of pou5f3 by combination of CRISPR/Cas9 and PGCT. A: Mutation efficiencies of gametes of each mutated positive F₀ adult fish (1♀, 9♂). B: The phenotypes of offspring crossed by female #1 and male #2 from sphere stage to 24 hpf. Note that the germ ring of mutant is thicker than the WT at shield stage, the epiboly is seriously affected during gastrulation, and a cluster of cells piles on the top of the dorsum (see LV, lateral view; DV, dorsal view) at 24 hpf. C: pou5f3 was barely expressed in mutants, compared with its high expression in WT. D: The expression of chd was expanded ventrally within the germ ring in mutants, compared with WT; eve1 was strongly reduced in mutants; sox32 and sox17, the markers for endoderm, were undetectable in mutant embryos; the expression of myoD was fuses ventrally in the mutant embryos; the expression of ntl was variably splited in mutant embryos, in comparison with its straight expression in the notochord in WT embryos.

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Fig. 5. Phenotypical analysis of MZchd generated by combination of CRISPR/Cas9 and PGCT. A: The fluorescent RNA in situ hybridization of chd on cryosections of zebrafish ovaries with antisense or sense chd RNA probes. B: RT-PCR analysis of chd during early development, β-actin was used as the internal control. C: Mutation efficiencies of gametes of each mutated positive F₀ adult fish (2♀, 6♂). D: Statistics of the phenotypes of F₁ offspring incrossed by two females (#1 and #2) and the male #1. C1 shows similar phenotype of WT, C2 shows smaller eyes and enlarged blood island, C3 shows severe head defects and tail blood island enlargement. E: The phenotypes of F₁ offspring incrossed by heterozygotes of chd mutants. C1 shows the WT like phenotype, C2 shows smaller eyes and enlarged blood island, a typical phenotype of zygotic mutant.

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Table 1. Positive rate of PGCT and the fertility of grown adults after PGCT.

Gene name	Test No.	Number of transplant	Number of survivors at 35 hpf	Number of successful PGCT embryos (%)	Number of survival adults	Number of fertile adults	Number of adults producing mutant embryo
tcf7l1a	1	64	30	7 (23.3)	7 (2♀5♂)	4 (2♀2♂)	4 (2♀2♂)
	2	66	37	9 (24.3)	6 (1♀5♂)	4 (1♀3♂)	4 (1♀3♂)
	3	62	31	8 (25.8)	5 (1♀4♂)	3 (1♀2♂)	3 (1♀2♂)
pou5f3	1	108	32	6 (18.7)	3 (1♀2♂)	1 (1♀0♂)	0
	2	97	45	11 (24.4)	8 (1♀7♂)	4 (1♀3♂)	3 (0♀3♂)
	3	113	63	15 (23.8)	13 (1♀12♂)	7 (1♀6♂)	7 (1♀6♂)
chd	1	72	32	9 (28.1)	7 (2♀5♂)	5 (2♀3♂)	3 (1♀2♂)
	2	56	34	10 (29.4)	6 (1♀5♂)	3 (1♀2♂)	1 (0♀1♂)
	3	67	38	10 (26.3)	8 (2♀6♂)	5 (2♀3♂)	4 (1♀3♂)

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参考文献(43)

[1]	Bassett, A.R., Tibbit, C., Ponting, C.P., Liu, J.L., 2013. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep 4, 220-228.
[2]	Bontems, F., Stein, A., Marlow, F., Lyautey, J., Gupta, T., Mullins, M.C., Dosch, R., 2009. Bucky ball organizes germ plasm assembly in zebrafish. Curr Biol 19, 414-422.
[3]	Branam, A.M., Hoffman, G.G., Pelegri, F., Greenspan, D.S., 2010. Zebrafish chordin-like and chordin are functionally redundant in regulating patterning of the dorsoventral axis. Dev Biol 341, 444-458.
[4]	Brion, F., Tyler, C.R., Palazzi, X., Laillet, B., Porcher, J.M., Garric, J., Flammarion, P., 2004. Impacts of 17 beta-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-, juvenile- and adult-life stages in zebrafish (Danio rerio). Aquat Toxicol.68, 193-217.
[5]	Burgess, S., Reim, G., Chen, W.B., Hopkins, N., Brand, M., 2002. The zebrafish spiel-ohne-grenzen (spg) gene encodes the POU domain protein Pou2 related to mammalian Oct4 and is essential for formation of the midbrain and hindbrain, and for pre-gastrula morphogenesis. Development 129, 905-916.
[6]	Chang, N., Sun, C., Gao, L., Zhu, D., Xu, X., Zhu, X., Xiong, J.W., Xi, J.J., 2013. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res 23, 465-472.
[7]	Ciruna, B., Weidinger, G., Knaut, H., Thisse, B., Thisse, C., Raz, E., Schier, A.F., 2002. Production of maternal-zygotic mutant zebrafish by germ-line replacement. Proc. Natl. Acad. Sci. U. S. A. 99, 14919-14924.
[8]	Dosch, R., Wagner, D.S., Mintzer, K.A., Runke, G., Wiemelt, A.P., Mullins, M.C., 2004. Maternal control of vertebrate development before the midblastula transition: mutants from the zebrafish I. Dev Cell 6, 771-780.
[9]	Gritsman, K., Zhang, J.J., Cheng, S., Heckscher, E., Talbot, W.S., Schier, A.F., 1999. The EGF-CFC protein one-eyed pinhead is essential for nodal signaling. Cell 97, 121-132.
[10]	Hammerschmidt, M., Pelegri, F., Mullins, M.C., Kane, D.A., van Eeden, F.J., Granato, M., Brand, M., Furutani-Seiki, M., Haffter, P., Heisenberg, C.P., Jiang, Y.J., Kelsh, R.N., Odenthal, J., Warga, R.M., Nusslein-Volhard, C., 1996. dino and mercedes, two genes regulating dorsal development in the zebrafish embryo. Development 123, 95-102.
[11]	Harvey, S.A., Sealy, I., Kettleborough, R., Fenyes, F., White, R., Stemple, D., Smith, J.C., 2013. Identification of the zebrafish maternal and paternal transcriptomes. Development 140, 2703-2710.
[12]	He, M.D., Zhang, F.H., Wang, H.L., Wang, H.P., Zhu, Z.Y., Sun, Y.H., 2015. Efficient ligase 3-dependent microhomology-mediated end joining repair of DNA double-strand breaks in zebrafish embryos. Mutat Res 780, 86-96.
[13]	Heyn, P., Kalinka, A.T., Tomancak, P., Neugebauer, K.M., 2015. Introns and gene expression: cellular constraints, transcriptional regulation, and evolutionary consequences. Bioessays 37, 148-154.
[14]	Hruscha, A., Krawitz, P., Rechenberg, A., Heinrich, V., Hecht, J., Haass, C., Schmid, B., 2013. Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. Development 140, 4982-4987.
[15]	Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M.L., Tsai, S.Q., Sander, J.D., Peterson, R.T., Yeh, J.R., Joung, J.K., 2013. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31, 227-229.
[16]	Kim, C.H., Oda, T., Itoh, M., Jiang, D., Artinger, K.B., Chandrasekharappa, S.C., Driever, W., Chitnis, A.B., 2000. Repressor activity of headless/Tcf3 is essential for vertebrate head formation. Nature 407, 913-916.
[17]	Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F., 1995. Stages of embryonic development of the zebrafish. Dev Dyn 203, 253-310.
[18]	Koprunner, M., Thisse, C., Thisse, B., Raz, E., 2001. A zebrafish nanos-related gene is essential for the development of primordial germ cells. Genes Dev. 15, 2877-2885.
[19]	Krishnakumar, P., Riemer, S., Perera, R., Lingner, T., Goloborodko, A., Khalifa, H., Bontems, F., Kaufholz, F., El-Brolosy, M.A., Dosch, R., 2018. Functional equivalence of germ plasm organizers. PLoS Genet 14, e1007696.
[20]	Lacerda, S., Costa, G., Campos-Junior, P., Segatelli, T., Yazawa, R., Takeuchi, Y., Morita, T., Yoshizaki, G., Franca, L., 2013. Germ cell transplantation as a potential biotechnological approach to fish reproduction. Fish Physiol Biochem 39, 3-11.
[21]	Lee, M.T., Bonneau, A.R., Takacs, C.M., Bazzini, A.A., DiVito, K.R., Fleming, E.S., Giraldez, A.J., 2013. Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition. Nature 503, 360-364.
[22]	Lewis, J.L., Bonner, J., Modrell, M., Ragland, J.W., Moon, R.T., Dorsky, R.I., Raible, D.W., 2004. Reiterated Wnt signaling during zebrafish neural crest development. Development 131, 1299-1308.
[23]	Liu, Y., Zhang, C., Zhang, Y., Lin, S., Shi, D.L., Shao, M., 2018. Highly efficient genome editing using oocyte-specific zcas9 transgenic zebrafish. J. Genet. Genomics. 45, 509-512.
[24]	Moreno-Mateos, M.A., Vejnar, C.E., Beaudoin, J.D., Fernandez, J.P., Mis, E.K., Khokha, M.K., Giraldez, A.J., 2015. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods 12, 982-988.
[25]	Nasevicius, A., Ekker, S.C., 2000. Effective targeted gene 'knockdown' in zebrafish. Nat. Genet. 26, 216-220.
[26]	Patton, E.E., Zon, L.I., 2001. The art and design of genetic screens: zebrafish. Nat Rev Genet 2, 956-966.
[27]	Reim, G., Brand, M., 2006. Maternal control of vertebrate dorsoventral axis formation and epiboly by the POU domain protein Spg/Pou2/Oct4. Development 133, 2757-2770.
[28]	Reim, G., Mizoguchi, T., Stainier, D.Y., Kikuchi, Y., Brand, M., 2004. The POU domain protein Spg (Pou2/Oct4) is essential for endoderm formation in cooperation with the HMG domain protein Casanova. Dev. Cell 6, 91-101.
[29]	Saito, T., Goto-Kazeto, R., Arai, K., Yamaha, E., 2008. Xenogenesis in teleost fish through generation of germ-line chimeras by single primordial germ cell transplantation. Biol Reprod 78, 159-166.
[30]	Schulte-Merker, S., Lee, K.J., McMahon, A.P., Hammerschmidt, M., 1997. The zebrafish organizer requires chordino. Nature 387, 862-863.
[31]	Sun, Y., Zhang, B., Luo, L., Shi, D.-L., Cui, Z., Huang, H., Cao, Y., Shu, X., Zhang, W., Zhou, J., Li, Y., Du, J., Zhao, Q., Chen, J., Zhong, H., Zhong, T.P., Li, L., Xiong, J.-W., Peng, J., Xiao, W., Zhang, J., Yao, J., Yin, Z., Mo, X., Peng, G., Zhu, J., Chen, Y., Zhou, Y., Liu, D., Pan, W., Zhang, Y., Ruan, H., Liu, F., Zhu, Z., Meng, A., 2020. Systematical genome editing of the genes in zebrafish Chromosome 1 by CRISPR/Cas9. Genome Research, 30: 118-126.
[32]	Sun, Y.H., 2017. Genome editing opens a new era for physiological study and directional breeding of fishes. Sci. Bull. 62, 157-158.
[33]	Thyme, S.B., Schier, A.F., 2016. Polq-Mediated End Joining Is Essential for Surviving DNA Double-Strand Breaks during Early Zebrafish Development (vol 15, pg 707, 2016). Cell Rep. 15, 1611-1613.
[34]	Tzung, K.W., Goto, R., Saju, J.M., Sreenivasan, R., Saito, T., Arai, K., Yamaha, E., Hossain, M.S., Calvert, M.E., Orban, L., 2015. Early depletion of primordial germ cells in zebrafish promotes testis formation. Stem Cell Rep. 5, 156.
[35]	Tzur, Y.B., Friedland, A.E., Nadarajan, S., Church, G.M., Calarco, J.A., Colaiacovo, M.P., 2013. Heritable custom genomic modifications in Caenorhabditis elegans via a CRISPR-Cas9 system. Genetics 195, 1181-1185.
[36]	Veil, M., Schaechtle, M.A., Gao, M., Kirner, V., Buryanova, L., Grethen, R., Onichtchouk, D., 2018. Maternal Nanog is required for zebrafish embryo architecture and for cell viability during gastrulation. Development 145.
[37]	Wagner, D.S., Dosch, R., Mintzer, K.A., Wiemelt, A.P., Mullins, M.C., 2004. Maternal control of development at the midblastula transition and beyond: mutants from the zebrafish II. Dev Cell 6, 781-790.
[38]	Wei, C.Y., Wang, H.P., Zhu, Z.Y., Sun, Y.H., 2014. Transcriptional factors smad1 and smad9 act redundantly to mediate zebrafish ventral specification downstream of smad5. J Biol Chem 289, 6604-6618.
[39]	Weidinger, G., Stebler, J., Slanchev, K., Dumstrei, K., Wise, C., Lovell-Badge, R., Thisse, C., Thisse, B., Raz, E., 2003. dead end, a novel vertebrate germ plasm component, is required for zebrafish primordial germ cell migration and survival. Cur. Biol. 13, 1429-1434.
[40]	Welten, M.C., de Haan, S.B., van den Boogert, N., Noordermeer, J.N., Lamers, G.E., Spaink, H.P., Meijer, A.H., Verbeek, F.J., 2006. ZebraFISH: fluorescent in situ hybridization protocol and three-dimensional imaging of gene expression patterns. Zebrafish 3, 465-476.
[41]	Xiong, F., Wei, Z.Q., Zhu, Z.Y., Sun, Y.H., 2013. Targeted expression in zebrafish primordial germ cells by Cre/loxP and Gal4/UAS systems. Mar Biotechnol (NY) 15, 526-539.
[42]	Ye, D., Zhu, L., Zhang, Q., Xiong, F., Wang, H., Wang, X., He, M., Zhu, Z., Sun, Y., 2019. Abundance of Early Embryonic Primordial Germ Cells Promotes Zebrafish Female Differentiation as Revealed by Lifetime Labeling of Germline. Mar Biotechnol (NY) 21, 217-228.
[43]	Zhang, F.H., Wang, H.P., Huang, S.Y., Xiong, F., Zhu, Z.Y., Sun, Y., 2016. A comparison of the knockout efficiencies of two codon-optimized Cas9 coding sequences in zebrafish embryos. Hereditas(Beijing) 38, 144-154 (in Chinese with an English abstract).