Abstract
Silkworm is a lepidopteran insect that has been used as a model for a wide variety of biological studies. The microinjection technique is available and it is possible to cause transgenesis as well as target gene disruption via the genome editing technique. TALEN-mediated knock-out is especially effective in this species. We also succeeded in the precise and efficient integration of a donor vector using the Precise Integration into Target Chromosome (PITCh) method. Here, we describe protocols for ZFN, TALEN, and CRISPR/Cas9-mediated genome editing as well as the PITCh technique in the silkworm. We consider that all of these techniques can contribute to the further promotion of various biological studies in the silkworm and other insect species.
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Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Kômoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol 18:81–84
The International Silkworm Genome Consortium (2008) The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect Biochem Mol Biol 38:1036–1045
Takasu Y, Kobayashi I, Beumer K, Uchino K, Sezutsu H, Sajwan S, Carroll D, Tamura T, Zurovec M (2010) Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection. Insect Biochem Mol Biol 40:759–765
Fujii T, Abe H, Katsuma S, Mita K, Shimada T (2008) Mapping of sex-linked genes onto the genome sequence using various aberrations of the Z chromosome in Bombyx mori. Insect Biochem Mol Biol 38:1072–1079
Sajwan S, Takasu Y, Tamura T, Uchino K, Sezutsu H, Zurovec M (2013) Efficient disruption of endogenous Bombyx gene by TAL effector nucleases. Insect Biochem Mol Biol 43:17–23
Takasu Y, Sajwan S, Daimon T, Osanai-Futahashi M, Uchino K, Sezutsu H, Tamura T, Zurovec M (2013) Efficient TALEN construction for Bombyx mori gene targeting. PLoS One 9:e73458
Yoda S, Yamaguchi J, Mita K, Yamamoto K, Banno Y, Ando T, Daimon T, Fujiwara H (2014) The transcription factor Apontic-like controls diverse colouration pattern in caterpillars. Nat Commun 5:4936
Daimon T, Uchibori M, Nakao H, Sezutsu H, Shinoda T (2015) Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle. Proc Natl Acad Sci U S A 112:E4226–E4235
Enya S, Daimon T, Igarashi F, Kataoka H, Uchibori M, Sezutsu H, Shinoda T, Niwa R (2015) The silkworm glutathione S-transferase gene noppera-bo is required for ecdysteroid biosynthesis and larval development. Insect Biochem Mol Biol 61:1–7
Osanai-Futahashi M, Tatematsu K, Futahashi R, Narukawa J, Takasu Y, Kayukawa T, Shinoda T, Ishige T, Yajima S, Tamura T, Yamamoto K, Sezutsu H (2015) Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis. Heredity 116:135–145
Shiomi K, Takasu Y, Kunii M, Tsuchiya R, Mukaida M, Kobayashi M, Sezutsu H, Ichida Takahama M, Mizoguchi A (2015) Disruption of diapause induction by TALEN-based gene mutagenesis in relation to a unique neuropeptide signaling pathway in Bombyx. Sci Rep 5:15566
Daimon T, Kiuchi T, Takasu Y (2014) Recent progress in genome engineering techniques in the silkworm, Bombyx mori. Develop Growth Differ 56:14–25
Wang Y, Tan A, Xu J, Li Z, Zeng B, Ling L, You L, Chen Y, James AA, Huang Y (2014) Site-specific, TALENs-mediated transformation of Bombyx mori. Insect Biochem Mol Biol 55:26–30
Nakade S, Tsubota T, Sakane Y, Kume S, Sakamoto N, Obara M, Daimon T, Sezutsu H, Yamamoto T, Sakuma T, Suzuki KT (2014) Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat Commun 5:5560
McVey M, Lee SE (2008) MMEJ repair of double-strand breaks (director’s cut): deleted sequences and alternative endings. Trends Genet 24:529–538
Sakuma T, Takenaga M, Kawabe Y, Nakamura T, Kamihira M, Yamamoto T (2015) Homologous recombination-independent large gene cassette knock-in in CHO cells using TALEN and MMEJ-directed donor plasmids. Int J Mol Sci 16:23849–23866
Xia Q, Cheng D, Duan J, Wang G, Cheng T, Zha X, Liu C, Zhao P, Dai F, Zhang Z, He N, Zhang L, Xiang Z (2007) Microarray-based gene expression profiles in multiple tissues of the domesticated silkworm, Bombyx mori. Genome Biol 8:R162
Tsubota T, Uchino K, Suzuki KT, Tanaka H, Kayukawa T, Shinoda T, Sezutsu H (2014) Identification of a novel strong and ubiquitous promoter/enhancer in the silkworm Bombyx mori. G3 (Bethesda) 4:1347–1357
Mandell JG, Barbas IIICF (2006) Zinc finger tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res 34:W516–W523
Carroll D, Morton JJ, Beumer KJ, Segal DJ (2006) Design, construction and in vitro testing of zinc finger nucleases. Nat Protoc 1:1329–1341
Takasu Y, Tamura T, Sajwan S, Kobayashi I, Zurovec M (2014) The use of TALENs for nonhomologous end joining mutagenesis in silkworm and fruitfly. Methods 69:46–57
Takasu Y, Tamura T, Goldsmith M, Zurovec M (2016) Targeted mutagenesis in Bombyx mori using TALENs. Methods Mol Biol 1338:127–142
Naito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31:1120–1123
Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh J-RJ, Joung JK (2013) Efficient genome editing in zabrafish using a CRISPR-Cas system. Nat Biotechnol 31:227–229
Sakuma T, Nakade S, Sakane Y, Suzuki KT, Yamamoto T (2016) MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nat Protoc 11:118–133
Thomas JL, Da Rocha M, Besse A, Mauchamp B, Chavancy G (2002) 3xP3-EGFP marker facilitates screening for transgenic silkworm Bombyx mori L. from the embryonic stage onwards. Insect Biochem Mol Biol 32:247–253
Masumoto M, Ohde T, Shiomi K, Yaginuma T, Niimi T (2012) A baculovirus immediate-early gene, ie1, promoter drives efficient expression of a transgene in both Drosophila melanogaster and Bombyx mori. PLoS One 7:e49323
Imamura M, Nakai J, Inoue S, Quan GX, Kanda T, Tamura T (2003) Targeted gene expresion using the GAL4/UAS system in the silkworm Bombyx mori. Genetics 165:1329–1340
Kobayashi I, Uchino K, Sezutsu H, Iizuka T, Tamura T (2007) Development of a new piggyBac vector for generating transgenic silkworms using the kynurenine 3-mono oxygenase gene. J Insect Biotechnol Sericol 76:145–148
Osanai-Futahashi M, Ohde T, Hirata J, Uchino K, Futahashi R, Tamura T, Niimi T, Sezutsu H (2012) A visible dominant marker for insect transgenesis. Nat Commun 3:1295
Acknowledgment
This work was supported by grants-in-aid from the Ministry of Agriculture, Forestry, and Fisheries.
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Tsubota, T., Sezutsu, H. (2017). Genome Editing of Silkworms. In: Hatada, I. (eds) Genome Editing in Animals. Methods in Molecular Biology, vol 1630. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7128-2_17
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DOI: https://doi.org/10.1007/978-1-4939-7128-2_17
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