The silkworm (Bombyx mori) is a lepidopteran model insect that has been utilized for basic research and industrial applications. In this species, transcription activator-like effector nucleases (TALENs) have been found to function efficiently, and we previously developed a TALEN-mediated genome editing system for knockout and knock-in experiments using plasmids and single-stranded oligodeoxynucleotides (ssODNs) as donors. By contrast, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing, especially for gene integration, remains limited. In this study, we attempted to improve CRISPR-Cas systems to expand the utility of genome editing in the silkworm. Codon optimization of Cas9 improved genome editing efficiency, and single-guide RNA utilization also resulted in a higher genome editing efficiency than crRNA/tracrRNA when Cas9 messenger RNA (mRNA) was used. CRISPR-Cas12a-mediated genome editing and targeted sequence integration using ssODNs were both successfully performed. Overall, our study provides a robust technical platform that can facilitate basic and applied silkworm studies.
Get full access to this article
View all access options for this article.
References
1.
TamuraT, ThibertC, RoyerC, et al.Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol, 2000; 18(1):81–84; doi: 10.1038/71978
2.
The International Silkworm Genome Consortium. The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect Biochem Mol Biol, 2008; 38(12):1036–1045; doi: 10.1016/j.ibmb.2008.11.004
3.
KuwanaY, SezutsuH, NakajimaK, et al.High-toughness silk produced by a transgenic silkworm expressing spider (Araneus ventricosus) dragline silk protein. PLoS One, 2014; 9(8):e105325; doi: 10.1371/journal.pone.0105325
4.
TatematsuKI, IkedaM, WakabayashiY, et al.Silkworm recombinant bovine zona pellucida protein 4 (bZP4) as a potential female immunocontraceptive antigen; impaired sperm-oocyte interaction and ovarian dysfunction. J Reprod Dev, 2021; 67(6):402–406; doi: 10.1262/jrd.2021-103
5.
TakasuY, KobayashiI, BeumerK, et al.Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection. Insect Biochem Mol Biol, 2010; 40(10):759–765; doi: 10.1016/j.ibmb.2010.07.012
6.
FujiiT, AbeH, KatsumaS, et al.Mapping of sex-linked genes onto the genome sequence using various aberrations of the Z chromosome in Bombyx mori. Insect Biochem Mol Biol, 2008; 38(12):1072–1079; doi: 10.1016/j.ibmb.2008.03.004
7.
TakasuY, SajwanS, DaimonT, et al.Efficient TALEN construction for Bombyx mori gene targeting. PLoS One, 2013; 8(9):e73458; doi: 10.1371/journal.pone.0073458
8.
NakadeS, TsubotaT, SakaneY, et al.Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat Commun, 2014; 5:5560; doi: 10.1038/ncomms6560
9.
TakasuY, KobayashiI, TamuraT, et al.Precise genome editing in the silkworm Bombyx mori using TALENs and ds- and ssDNA donors - A practical approach. Insect Biochem Mol Biol, 2016; 78:29–38; doi: 10.1016/j.ibmb.2016.08.006
10.
TakasuY, YamadaN, KojimaK, et al.Fibroin heavy chain gene replacement with a highly ordered synthetic repeat sequence in Bombyx mori. Insect Biochem Mol Biol, 2023; 161:104002; doi: 10.1016/j.ibmb.2023.104002
11.
DaimonT, KiuchiT, TakasuY. Recent progress in genome engineering techniques in the silkworm, Bombyx mori. Dev Growth Differ, 2014; 56(1):14–25; doi: 10.1111/dgd.12096
12.
ZengB, ZhanS, WangY, et al.Expansion of CRISPR targeting sites in Bombyx mori. Insect Biochem Mol Biol, 2016; 72:31–40; doi: 10.1016/j.ibmb.2016.03.006
13.
ZouY, YeA, LiuS, et al.Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori. BMC Biotechnol, 2021; 21(1):54; doi: 10.1186/s12896-021-00714-6
14.
SunL, ZhangT, LanX, et al.High-throughput screening of PAM-flexible Cas9 variants for expanded genome editing in the silkworm (Bombyx mori). Insects, 2024; 15(4):241; doi: 10.3390/insects15040241
15.
LiuY, MaS, ChangJ, et al.Tissue-specific genome editing of laminA/C in the posterior silk glands of Bombyx mori. J Genet Genomics, 2017; 44(9):451–459; doi: 10.1016/j.jgg.2017.09.003
16.
XuJ, ChenRM, ChenSQ, et al.Identification of a germline-expression promoter for genome editing in Bombyx mori. Insect Sci, 2019; 26(6):991–999; doi: 10.1111/1744-7917.12657
17.
WangY, DuT, LiA, et al.Establishment and application of a silkworm CRISPR/Cas9 tool for conditionally manipulating gene disruption in the epidermis. Insect Biochem Mol Biol, 2022; 151:103861; doi: 10.1016/j.ibmb.2022.103861
18.
ZhangX, XiaL, DayBA, et al.CRISPR/Cas9 initiated transgenic silkworms as a natural spinner of spider silk. Biomacromolecules, 2019; 20(6):2252–2264; doi: 10.1021/acs.biomac.9b00193
19.
HwangWY, FuY, ReyonD, et al.Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol, 2013; 31(3):227–229; doi: 10.1038/nbt.2501
20.
KistlerKE, VosshallLB, MatthewsBJ. Genome-engineering with CRISPR-Cas9 in the mosquito Aedes aegypti. Cell Rep, 2015; 11(1):51–60; doi: 10.1016/j.celrep.2015.03.009
XingHL, DongL, WangZP, et al.A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol, 2014; 14:327; doi: 10.1186/s12870-014-0327-y
23.
LeeK, ZhangY, KleinstiverBP, et al.Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize. Plant Biotechnol J, 2019; 17(2):362–372; doi: 10.1111/pbi.12982
24.
LiJ, ZhangS, ZhangR, et al.Efficient multiplex genome editing by CRISPR/Cas9 in common wheat. Plant Biotechnol J, 2021; 19(3):427–429; doi: 10.1111/pbi.13508
25.
LiG, SretenovicS, EisensteinE, et al.Highly efficient C-to-T and A-to-G base editing in a Populus hybrid. Plant Biotechnol J, 2021; 19(6):1086–1088; doi: 10.1111/pbi.13581
26.
RenQ, SretenovicS, LiuG, et al.Improved plant cytosine base editors with high editing activity, purity, and specificity. Plant Biotechnol J, 2021; 19(10):2052–2068; doi: 10.1111/pbi.13635
27.
RandallLB, SretenovicS, WuY, et al.Genome- and transcriptome-wide off-target analyses of an improved cytosine base editor. Plant Physiol, 2021; 187(1):73–87; doi: 10.1093/plphys/kiab264
28.
SwartsDC, JinekM. Cas9 versus Cas12a/Cpf1: Structure-function comparisons and implications for genome editing. Wiley Interdiscip Rev RNA, 2018; 9(5):e1481; doi: 10.1002/wrna.1481
29.
ZetscheB, GootenbergJS, AbudayyehOO, et al.Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 2015; 163(3):759–771; doi: 10.1016/j.cell.2015.09.038
MaS, LiuY, LiuY, et al.An integrated CRISPR Bombyx mori genome editing system with improved efficiency and expanded target sites. Insect Biochem Mol Biol, 2017; 83:13–20; doi: 10.1016/j.ibmb.2017.02.003
32.
KômotoN, QuanGX, SezutsuH, et al.A single-base deletion in an ABC transporter gene causes white eyes, white eggs, and translucent larval skin in the silkworm w-3oe mutant. Insect Biochem Mol Biol, 2009; 39(2):152–156; doi: 10.1016/j.ibmb.2008.10.003
33.
ThorpeHM, SmithMC. In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase invertase family. Proc Natl Acad Sci U S A, 1998; 95(10):5505–5510; doi: 10.1073/pnas.95.10.5505
34.
TatematsuK, YamamotoK, UchinoK, et al.Positional cloning of silkworm white egg 2 (w-2) locus shows functional conservation and diversification of ABC transporters for pigmentation in insects. Genes Cells, 2011; 16(4):331–342; doi: 10.1111/j.1365-2443.2011.01490.x
35.
KelleyML, StrezoskaŽ, HeK, et al.Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing. J Biotechnol, 2016; 233:74–83; doi: 10.1016/j.jbiotec.2016.06.011
36.
NagyL, RiddifordL, KiguchiK. Morphogenesis in the early embryo of the lepidopteran Bombyx mori. Dev Biol, 1994; 165(1):137–151; doi: 10.1006/dbio.1994.1241
37.
SungYH, KimJM, KimH-T, et al.Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res, 2014; 24(1):125–131; doi: 10.1101/gr.163394.113
38.
HuP, ZhaoX, ZhangQ, et al.Comparison of various nuclear localization signal-fused Cas9 proteins and Cas9 mRNA for genome editing in zebrafish. G3 (Bethesda), 2018; 8(3):823–831; doi: 10.1534/g3.117.300359
39.
WaizumiR, HirayamaC, TomitaS, et al.A major endogenous glycoside hydrolase mediating quercetin uptake in Bombyx mori. PLoS Genet, 2024; 20(1):e1011118; doi: 10.1371/journal.pgen.1011118
40.
ZhangZ, NiuB, JiD, et al.Silkworm genetic sexing through W chromosome linked, targeted gene integration. Proc Natl Acad Sci U S A, 2018; 115(35):8752–8756; doi: 10.1073/pnas.1810945115
41.
YonemuraN, TamuraT, UchinoK, et al.phiC31-integrase-mediated, site-specific integration of transgenes in the silkworm, Bombyx mori (Lepidoptera: Bombycidae). Appl Entomol Zool, 2013; 48(3):265–273; doi: 10.1007/s13355-013-0182-6
42.
SuzukiTK, KoshikawaS, KobayashiI, et al.Modular cis-regulatory logic of yellow gene expression in silkmoth larvae. Insect Mol Biol, 2019; 28(4):568–577; doi: 10.1111/imb.12574
43.
YoshimiK, KunihiroY, KanekoT, et al.ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes. Nat Commun, 2016; 7:10431; doi: 10.1038/ncomms10431
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
