Pan-Genomics and CRISPR genome editing for silk biosynthesis improvement in Bombyx mori: A Review

Introduction

Sericulture, the agricultural practice centered on the mulberry silkworm, Bombyx mori, has been a globally significant industry for millennia, playing a critical role in textile production and supporting numerous livelihoods, particularly across Asia (Bora et al., 2025) [3] . Silk, often referred to as "The Queen of Textiles," remains a highly sought-after natural fibre due to its exceptional quality, durability, and aesthetic appeal, factors that make its yield and intrinsic properties economically critical (Pavithra et al., 2024; Sree et al., 2024) [27] . The global sericulture industry, particularly in China, is valued at tens of billions of CNY annually, highlighting its substantial economic impact (Lu et al., 2026) [36] . Traditionally, enhancing silk yield and quality has relied heavily on conventional breeding methods. However, these methods face considerable limitations, including susceptibility to climate variability, outbreaks of pathogens, and inherent genetic constraints within domesticated silkworm strains (Bora et al., 2025) [3] . For instance, silkworm health is frequently compromised by a range of pathogens, such as viruses, bacteria, fungi, and protozoa, which can lead to significant productivity losses. The Bombyx mori nucleopolyhedrovirus (BmNPV) alone is a major concern, often accounting for more than 60% of total losses from silkworm diseases (Kaushik et al., 2026) [13] . Furthermore, environmental stressors like temperature fluctuations and pesticide exposure can directly impact cocoon production and silk quality, posing ongoing challenges to the industry (Lu et al., 2026; Kaushik et al., 2026) [13] . The laborious task of separating male and female pupae for egg production in grainage also highlights a practical limitation in conventional sericulture, especially given the typically lower fecundity of sex-limited breeds (Kasa et al., 2023) [12] . The emergence of advanced molecular biology tools, specifically pan-genomics and genome editing technologies like CRISPR-Cas systems, has revolutionized silkworm research, offering unprecedented opportunities for genetic improvement and sustainable silk production (Bora et al., 2025; Kalpana et al., 2024) [3,35] . Bombyx mori is a critical lepidopteran model organism, not only for sericulture research but also for broader biological studies, owing to its well-characterized biology and significant economic value (Lu et al., 2023; Tong et al., 2023) [43] . Its long history of domestication has resulted in extreme domestication bottlenecks, leading to substantial structural and presence-absence variation (PAV) within its populations (Tong et al., 2023) [43] . This considerable genetic diversity necessitates pan-genomic approaches to comprehensively capture the species genomic repertoire, moving beyond single reference genomes to understand the full spectrum of genetic variation across different strains (Lu et al., 2023; Tong et al., 2022) [43,22] . Recent efforts have led to the assembly of high-resolution pan-genomes for B. mori using long-read sequencing technologies, which have revealed thousands of novel genes and regulatory elements previously absent from earlier reference genomes (Tong et al., 2022) [43] . For example, the SilkMeta platform provides a comprehensive database integrating pan-genomic and multi-omic data from 1082 short-read and 546 long-read assembled genomes, serving as a valuable resource for genotype-phenotype association studies (Lu et al., 2023) [22] . This extensive genomic information, combined with the precision of CRISPR-Cas9 genome editing, facilitates targeted modifications to key genes involved in silk biosynthesis and other economically important traits (Bora et al., 2025; Pavithra et al., 2024) [3,27] . For instance, the BmSur8 gene, which encodes a scaffold protein, has been identified as a positive regulator of the RAS-MAPK signaling cascade, significantly influencing cocoon silk production (Zhou et al., 2025) [53] . The application of nutrient supplements, such as spermidine, has also shown promise in improving fecundity and economic traits in bivoltine cocoon color sex-limited breeds, addressing previous limitations in these strains (Sree et al., 2024; Kasa et al., 2023) [12,36] . CRISPR-Cas systems represent a powerful genome editing technology that enables precise alterations to the genetic code of living organisms (Bora et al., 2025) [3] . In Bombyx mori, this technology is being applied to enhance various aspects of sericulture, including improving silk yield and quality, increasing disease resistance, and facilitating sex control for optimized production (Bora et al., 2025; Pavithra et al., 2024) [3,27] . The integration of these biotechnological advancements, ranging from comprehensive genomic understanding to precise genetic engineering, signifies a paradigm shift towards functionally guided engineering of silk traits, offering new hope for addressing the persistent challenges faced by the sericulture industry (Bora et al., 2025; Kalpana et al., 2024) [3,35] .

Silk biosynthesis in Bombyx mori: Silk biosynthesis in

Bombyx mori occurs in the larval silk gland, a specialized organ responsible for producing silk proteins during the final larval stage (Xia et al., 2004). The major structural component of silk, fibroin, is synthesized in the posterior silk gland and consists mainly of fibroin heavy chain, light chain, and P25 proteins that form a stable complex (Inoue et al., 2000) [11] . Another important protein, sericin, is produced in the middle silk gland and acts as a binding material that coats fibroin fibers during cocoon formation (Kawasaki et al., 2007). The coordinated expression of silk protein genes and their secretion through the silk gland enable the formation of strong and continuous silk filaments used to construct the cocoon (Xia et al., 2004) [44] . [11,52,53] . Signalling protein Regulates silk yield through MAPK pathway

Gene Function Role in Silk Production

Pan-Genomics of Bombyx mori

Pan-genome studies in silkworm are redefining how silk yield and quality genes are discovered, especially by revealing structural variation and dispensable genes missed by single references. These genomic resources now directly connect to CRISPR-based functional validation and breeding for improved silk production. [38,42,44] Omics

Concept of Pan-Genome in insects

In insects, the pan-genome includes all genes present across a species, partitioned into core genes (present in almost all individuals) and dispensable/variable genes (present in subsets, often linked to adaptation) ( [42] . Comparable insect pan-genomes (Drosophila, butterflies, Apis cerana) show that SV-rich dispensable genomes strongly affect ecological adaptation and complex traits (Tong et al., 2023; li et al., 2024; Tong et al., 2021) [43] . [7] . Pan-genome scans identify hundreds of genes and SVs associated with domestication and breeding, including loci affecting behaviour, diapause, coloration, and silk traits (Tong et al., 2022; Tong et al., 2021) [42] . QTL mapping in crosses between B. mori and B. mandarina detected 11 cocoon yield QTLs, including one yield-enhancing allele contributed by the wild species and candidate genes (e.g., KWMTBOMO04917, KWMTBOMO12906) expressed in silk gland (Fang et al., 2020) [7] .

Comparative genomics of wild vs domesticated silkworm

Structural variations influencing silk traits

Structural variations (SVs) are pivotal drivers of phenotypic diversity in Bombyx mori, profoundly influencing silk traits through mechanisms such as copy number variations (CNVs), presence/absence variations (PAVs), and gene duplications within critical fibroin gene clusters (Tong et al., 2022; Fang et al., 2020) [7] . CNVs, which involve gains or losses of DNA segments, alter gene dosage and expression, thereby affecting the quantity of silk protein produced (Luan et al., 2021) [25] . Similarly, PAVs represent the complete presence or absence of genes or genomic regions, contributing unique physiological or biochemical pathways that directly impact silk quality and quantity across different silkworm strains (Tong et al., 2022) [42] . Gene duplications, particularly within the Fibroin Heavy (Fib-H), Fibroin Light (Fib-L), and P25 clusters, are directly correlated with increased silk yield and enhanced Fiber properties by augmenting the templates available for silk protein synthesis (Luan et al., 2021) [25] . The comprehensive functional annotation of novel pan-genes, identified through high-resolution pan-genomic studies, further elucidates the genetic architecture underlying silk production (Tong et al., 2022) [42] . Many of these pan genes are associated with traits targeted during artificial selection, particularly silk yield. Candidate genes such as Vps13d, which plays an important role in silk production, along with other genes involved in protein secretion, chaperone assisted protein folding, and transcriptional regulation of silk gland development, contribute significantly to variations in silk productivity. (Luan et al., [25] . Beyond protein-coding genes, the diversity in non-coding regulatory sequences, including promoters, enhancers, and microRNAs like let-7, plays a crucial role in fine-tuning gene expression, thus modulating the timing, location, and magnitude of gene activity that ultimately impacts silk production efficiency (Tong et al 2022; li et al., 2024) [42] .

Functional Annotation of Novel Pan-Genes

Pan-genomic analysis in Bombyx mori has identified over 7,000 novel genes and millions of structural variants, many under strong artificial selection for high silk yield and improved fibre fineness, thereby defining a rich pool of silkassociated pan-genes (Tong et al., 2022) [42] . Multi-omics and SNP-chip studies further refine this space, linking silk gland size, metabolism and cocoon traits to specific genes such as BmOCIAD1, BmCOPB2, Vps13d, BmSur8 and others, whose CRISPR perturbation substantially alters cocoon shell weight and silk output (Zhou et [26,38,53] . At the regulatory level, genome-wide maps of open chromatin and transcription factor (TF) binding in the silk gland identify tens of thousands of OCRs and hundreds of TF motifs, including CORE clusters that synergistically control silk-protein loci (Zhang et [26,38,42,[48][49][50][51] .

CRISPR-Based genome engineering in Bombyx mori

Pan-genome and SNP-array resources in Bombyx mori are now systematically annotated using silk-gland transcriptomes, QTL mapping, and association analyses to define candidate pan-genes for silk yield and quality (Kawamoto et al., 2019; Lu et al., 2021) [15] . Key yield-related loci include genes detected in cocoon yield QTLs and high-density SNP-array studies, for which CRISPR/Cas9 knockouts have validated causal roles in cocoon shell ratio and silk output (Zhang et al., 2018; Li et al., 2022). Functional studies highlight regulators such as BmVps13d and signalling components whose disruption reduces silk yield by altering energy and material metabolism in the silk gland (Zhao et al., 2019) [52] . Beyond structural silk-protein loci, genome editing of regulatory network genes like the circadian regulator Clock (Clk) has increased silk production, illustrating that systemic regulators outside fibroin clusters can be engineered for productivity gains (Xu et al., 2020) [45] . Regulatory sequence diversity is being captured with ATAC-seq and small-RNA profiling in B. mori and its wild ancestor B. mandarina, providing maps of open chromatin and small-RNA-regulated regions that guide precise CRISPR-mediated editing of promoters and enhancers for silk biosynthesis rewiring (Su et al., 2021). [41,45,30] [32] . These empirical networks can then be integrated into systems biology models, including pathway reconstruction for nitrogen and amino acid mobilization and genome-scale representations of silk gland metabolism, enabling in silico perturbation and metabolic flux analysis to predict how changes in key enzymes or alter fibroin/sericin synthesis and gland physiology (Yi et [2,46,53] .

Application

Integrating Pan-Genomics with CRISPR for Trait

Improvement: High-resolution silkworm pan-genomes now provide a catalogue of genes, structural variants, and domestication sweeps associated with silk yield, fineness, diapause, and other adaptive traits, enabling identification of high-value targets (e.g., BmChit β-GlcNAcase, silk-yield and fineness loci) for functional validation and editing (Tong et al., 2022) [42] . These pan-genomic signals, together with QTLs and GWAS hits discovered using tools such as the Silk_40K SNP array, prioritize candidate genes like BmOCIAD1 and BmCOPB2 that have been experimentally confirmed to increase cocoon shell weight and shell ratio when disrupted by CRISPR (Li et al., 2025). Once targets are defined, precision editing of elite commercial lines using optimized and broadened CRISPR platforms (integrated SpCas9/SaCas9/Cas12a systems, PAM-flexible SpCas9-NG/xCas9 variants, and improved delivery workflows) allows direct introduction of beneficial allelessuch as edits in BmEcKL1, let-7, or Clock that enlarge silk glands, extend growth, or shift physiology to increase silk output without losing complex backgrounds that underpin field performance (Ma et [4,39] . Finally, marker-assisted selection and genomic selection can be tightly integrated with genome editing: pan-genomeanchored SNP chips support genomic prediction of complex traits and tracking of edited alleles across breeding cycles, creating a feedback loop where MAS/GS identify superior haplotypes, CRISPR recreates or refines them in elite germplasm, and subsequent genomic evaluation quantifies gains in silk yield, quality, and resilience (Li et al., 2025; Bora et al., 2025; Ma et al., 2018) [3] .

Biosafety, Ethical and Regulatory Considerations

Rewiring silk biosynthesis in Bombyx mori with pan-genomics and CRISPR demands a rigorous treatment of biosafety, ethics and regulation, beginning with off-target effects at both the molecular and ecological levels. At the genome-scale, unintended mutations, chromosomal rearrangements, and large on-target indels remain key concerns, requiring careful sgRNA design, in silico off-target prediction, and whole-genome sequencing-based validation to minimize and monitor genomic irregularities in edited silkworm lines (Manghwar et al., 2020; Guo et al., 2023; Kawall et al., 2020) [9,14,31] . Beyond individual animals, ecological risk assessment must consider accidental escape of edited strains and, in future, gene-drive-based sericulture strategies, where self-propagating constructs might spread into wild Bombyx relatives, alter food webs, or affect non-target species through horizontal transfer or trophic interactions (Devos et [10,16,17] . Lessons from gene-drive work in mosquitoes and conceptual models for pest control emphasize phased testing, confinement, robust problem formulation, and pre-release modelling combined with post-release monitoring to manage uncertainty and avoid irreversible ecological impacts (Tajudeen et [1,40] . Finally, regulatory frameworks for edited insects are still evolving: in most jurisdictions, CRISPR-modified insects (with or without transgenes) fall under GMO or "new breeding technique" rules, but current guidance largely developed for plants or conventional GM animals has been judged adequate yet insufficient for insects containing engineered gene drives, prompting calls in the EU and elsewhere for expanded molecular characterization requirements, broader environmental risk assessment, and adaptive, stepwise authorization schemes that explicitly integrate ethical analysis, stakeholder engagement, and comparison with alternative pest-and disease-control options (Zhang et [14,41] .

Future Perspectives

Future research on pan-genomics and CRISPR-mediated rewiring of silk biosynthesis in Bombyx mori will focus on identifying novel alleles and regulatory genes associated with silk production and environmental adaptability. Integration of these genomic insights with AI-guided genome design may help predict key targets for precise genome editing. CRISPR technologies could also facilitate the development of climate-resilient silkworm strains capable of maintaining productivity under changing environmental conditions. In addition, digital sericulture platforms supported by data analytics and smart monitoring systems may link genomic innovations with improved rearing and management practices.

Conclusions

The integration of pan-genomics and CRISPR-mediated genome engineering is transforming research and innovation in Bombyx mori. Pan-genomic studies have revealed extensive structural variation, novel genes, and regulatory elements associated with silk production, providing a comprehensive view of the genetic architecture underlying economically important traits. Coupled with CRISPR-based functional validation, these genomic resources enable precise modification of key genes and regulatory networks controlling silk biosynthesis, cocoon yield, and fibre quality. Advances in systems biology and synthetic biology further support the rational rewiring of metabolic and regulatory pathways within the silk gland. Together, these approaches establish a powerful framework for accelerating genetic improvement in silkworms. The continued integration of genomic data, genome editing, and advanced breeding strategies will play a critical role in enhancing silk productivity, resilience, and sustainability, ultimately driving the modernization of the global sericulture industry.

Disclaimer (Artificial intelligence)

Author(s) hereby declares that NO generative AI technologies such as Large Language Mode (Chat GPT, COPILOT, etc) and text-to-image generators have been used during writing or editing of this manuscript.

Competing interests

Authors have declared that no competing interests exist.

References

1. Aslam M, Akram A, Iqbal M, Amber M, Amin J, Fatima S. From lab to field: CRISPR-based gene drives for disease control and prevention: An updated review. Planta Animalia. 2025;1(1):1-18.
2. Bashor CJ, Collins JJ. Understanding biological regulation through synthetic biology. Annu Rev Biophys. 2018;47(1):399-423.
3. Bora S, Narzary PR, Ahmed MH, Chetia P, Thengal AN. Integrating multi-omics, bioinformatics and genome editing for sustainable sericulture. Arch Curr Res Int. 2025;25(9):554-569. DOI:10.9734/acri/2025/v25i91519.
4. Brady D, Saviane A, Cappellozza S, Sandrelli F. An efficient workflow for screening and stabilizing CRISPR/Cas9-mediated mutant lines in Bombyx mori. Methods Protoc. 2020;4(1):4.
5. Devos Y, Mumford JD, Bonsall MB, Camargo AM, Firbank LG, Glandorf DC, et al. Potential use of gene drive modified insects against disease vectors, agricultural pests and invasive species poses new challenges for risk assessment. Crit Rev Biotechnol. 2022;42(2):254-270.
6. Devos Y, Mumford JD, Bonsall MB, Glandorf DC, Quemada HD. Risk management recommendations for environmental releases of gene drive modified insects. Biotechnol Adv. 2022;54:107807.
7. Fang SM, Zhou QZ, Yu QY, Zhang Z. Genetic and genomic analysis for cocoon yield traits in silkworm. Sci Rep. 2020;10(1):5682.
8. Grimes T, Potter SS, Datta S. Integrating gene regulatory pathways into differential network analysis of gene expression data. Sci Rep. 2019;9(1):5479.
9. Guo C, Ma X, Gao F, Guo Y. Off-target effects in CRISPR/Cas9 gene editing. Front Bioeng Biotechnol. 2023;11:1143157.
10. Hoepers AM, Heinemann JA, Zanatta CB, Chu P, Hiscox TC, Agapito-Tenfen SZ. Predicted multispecies unintended effects from outdoor genome editing. Ecotoxicol Environ Saf. 2024;282:116707.
11. Inoue S, Tanaka K, Arisaka F, Kimura S, Ohtomo K, Mizuno S. Silk fibroin of Bombyx mori is secreted assembling a high molecular mass elementary unit consisting of H-chain, L-chain and P25 with a 6:6:1 molar ratio. J Biol Chem. 2000;275(51):40517-40528. DOI:10.1074/jbc.M006897200.
12. Kasa M, Jolapuram S, Lima A, Didugu BGL, Poosapati JR, Mamillapalli A. Bivoltine cocoon color sex-limited breeds of Bombyx mori show enhanced economic performance and fecundity following spermidine supplementation. J Econ Entomol. 2023;116(5):16791688. DOI:10.1093/jee/toad126.
13. Kaushik A, Narzary PR, Ahmed MH, Saikia S, Thengal AN. The mulberry-silkworm nexus: Nutritional ecology, stress responses and their impact on silk production. Uttar Pradesh J Zool. 2025;46(18):99-109.
14. Kawall K, Cotter J, Then C. Broadening the GMO risk assessment in the EU for genome editing technologies in agriculture. Environ Sci Eur. 2020;32(1):106.
15. Kawamoto M, Jouraku A, Toyoda A, Yokoi K, Minakuchi Y, Katsuma S, et al. High-quality genome assembly of the silkworm reveals extensive gene duplication and structural variation. Insect Biochem Mol Biol. 2019;107:53-62. DOI:10.1016/j.ibmb.2019.02.007.
16. Komal J, Desai HR, Samal I, Mastinu A, Patel RD, Kumar PD, et al. Unveiling the genetic symphony: Harnessing CRISPR-Cas genome editing for effective insect pest management. Plants. 2023;12(23):3961.
17. Kumari P, Verma T, Yadav M, Pawar P, Mounika J, Singh B. Advancements in genome editing for insect control: A comprehensive review. Environ Ecol. 2024;42(1A):307-315.
18. Li C, Tong X, Gao R, Jiang J, Hu D, Zhou A, et al. Silk_40K SNP array facilitates the genomic prediction of complex traits in silkworm (Bombyx mori). J Adv Res. 2025. DOI:10.1016/j.jare.2025.11.021.
19. Li Y, Yao J, Sang H, Wang Q, Su L, Zhao X, et al. Pangenome analysis highlights the role of structural variation in the evolution and environmental adaptation of Asian honeybees. Mol Ecol Resour. 2024;24(2):e13905.
20. Li Z, Zhang X, Wang Y, Chen Q, Liu S, Xiang H. CRISPR/Cas9 mediated knockout of silk yield candidate genes improves understanding of cocoon shell ratio in Bombyx mori. Insect Sci. 2022;29(4):1021-1034. DOI:10.1111/1744-7917.12921.
21. Lu H, Wang J, Li F, Li B, Zhang Z. Genome-wide association and QTL mapping identify candidate genes for cocoon and silk traits in silkworm Bombyx mori. BMC Genomics. 2021;22:315. DOI:10.1186/s12864021-07616-3.
22. Lu K, Pan Y, Shen J, Yang L, Zhan C, Liang S, et al. SilkMeta: A comprehensive platform for sharing and exploiting pan-genomic and multi-omic silkworm data. Nucleic Acids Res. 2023;52(D1):D1024-D1032. DOI:10.1093/nar/gkad95.
23. Lu K, Zhan C, Shen J, Zhi C, Deng J, Lai K, et al. Characterization of spectrin family genes and their evolutionary roles in domestication and breeding of the silkworm Bombyx mori. Insects. 2025;16(6):556.
24. Lu ZP, Jiang JW, Zheng KY, Shao ZM, Wang XY, Li MW. Advances in resistance mechanisms and prevention strategies for pesticide poisoning in silkworms Bombyx mori. J Appl Entomol. 2026.
25. Luan Y, Li C, Liu Y, Zhang B, Gao R, Zuo W, et al. A cluster of genes at the terminal region of chromosome 1 contributes to silk yield in Bombyx mori. Res Square. 2021. DOI:10.21203/rs.3.rs-1031814/v1.
26. Luan Y, Zuo W, Li C, Gao R, Zhang H, Tong X, et al. Identification of genes that control silk yield by RNA sequencing analysis of silkworm strains of variable silk yield. Int J Mol Sci. 2018;19:3718. DOI:10.3390/ijms19123718.
27. Pavithra MR, Karur AS, Teja KSS, Parmar S, Sujatha GS, Kumar GA, et al. Genetic improvements in silkworms: Enhancing silk yield and quality. Uttar Pradesh J Zool. 2024;45(16):164-172.
28. Ma S, Liu Y, Liu Y, Chang J, Zhang T, Wang X, 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.
29. Ma S, Smagghe G, Xia Q. Genome editing in Bombyx mori: New opportunities for silkworm functional genomics and the sericulture industry. Insect Sci. 2018;25(3):423-435.
30. Ma Y, Zeng W, Ba Y, Luo Q, Ou Y, Liu R, et al. A single-cell transcriptomic atlas characterizes the silkproducing organ in the silkworm. Nat Commun. 2022;13(1):3316.
31. Manghwar H, Li B, Ding X, Hussain A, Lindsey K, Zhang X, Jin S. CRISPR/Cas systems in genome editing: Methodologies and tools for sgRNA design, off-target evaluation and strategies to mitigate off-target effects. Adv Sci. 2020;7(6):1902312.
32. Masuoka Y, Cao W, Jouraku A, Sakai H, Sezutsu H, Yokoi K. Co-expression network and time-course expression analyses to identify silk protein regulatory factors in Bombyx mori. Insects. 2022;13(2):131.
33. EFSA Panel on Genetically Modified Organisms (GMO), Naegeli H, Bresson JL, Dalmay T, Dewhurst IC, Epstein MM, et al. Adequacy and sufficiency evaluation of existing EFSA guidelines for the molecular characterisation, environmental risk assessment and post-market environmental monitoring of genetically modified insects containing engineered gene drives. EFSA J. 2020;18(11):e06297.
34. Pio G, Mignone P, Magazzù G, Zampieri G, Ceci M, Angione C. Integrating genome-scale metabolic modelling and transfer learning for human gene regulatory network reconstruction. Bioinformatics. 2022;38(2):487-493.
35. Kalpana R, Mithilasri M, Karthick Mani Bharathi B, Susikaran S, Sabarish M, Parthiban KT. Bridging tradition with innovation: Revolutionizing sericulture through biotechnological advancements. Int J Zool Appl Biosci. 2024;9(4):78-82. DOI:10.55126/ijzab.2024.v09.i04.013.
36. Sree KSS, Rani AS, Savithri G. Effect of nutrient supplements on economic traits of silkworm Bombyx mori L: A review. Uttar Pradesh J Zool. 2024;45(21):324-332. DOI:10.56557/upjoz/2024/v45i214642.
37. Su J, Li C, Wang L, Yu Y, Xia Q. Chromatin accessibility and small RNA landscapes in Bombyx mori and Bombyx mandarina reveal regulatory elements for silk gland function. Insect Mol Biol. 2021;30(5):422-436. DOI:10.1111/imb.12722.
38. Sun L, Sun B, Chen L, Ge Q, Chen K. Identification of genes associated with silk gland size using multi-omics in silkworm Bombyx mori. Insect Mol Biol. 2023;33:116. DOI:10.1111/imb.12870.
39. Sun L, Zhang T, Lan X, Zhang N, Wang R, Ma S, et al. High-throughput screening of PAM-flexible Cas9 variants for expanded genome editing in the silkworm Bombyx mori. Insects. 2024;15(4):241.
40. Tajudeen YA, Oladipo HJ, Oladunjoye IO, Oladipo MK, Shittu HD, Abdulmumeen IF, et al. Transforming malaria prevention and control: The prospects and challenges of gene drive technology for mosquito management. Ann Med. 2023;55(2):2302504.
41. Taning CNT, Van Eynde B, Yu N, Ma S, Smagghe G. CRISPR/Cas9 in insects: Applications, best practices and biosafety concerns. J Insect Physiol. 2017;98:245257.
42. Tong X, Han MJ, Lu K, Tai S, Liang S, Liu Y, et al. High-resolution silkworm pan-genome provides genetic insights into artificial selection and ecological adaptation. Nat Commun. 2022;13(1):5619.
43. Tong X, Han MJ, Lu K, Tai S, Liang S, Liu Y, et al. Reference genomes of 545 silkworms enable highthroughput exploring genotype-phenotype relationships. bioRxiv. 2021.
44. Xia Q, Zhou Z, Lu C, Cheng D, Dai F, Li B, et al. A draft sequence for the genome of the domesticated silkworm Bombyx mori. Science. 2004;306(5703):1937-1940.
45. Xu H, Liu Y, Zhang T, Chen P, Li M. Editing the circadian gene Clock increases silk production in Bombyx mori via CRISPR/Cas9. Insect Biochem Mol Biol. 2020;125:103430.
46. Yang S, He M, Li X, Dong H, Lei H, Wang F, et al. Knocked-out Bombyx mori protein disulfide isomerase decreases silk yields and mechanical properties by affecting post-translational modification of silk proteins. Insects. 2025;16(7):684.
47. Yi MY, Yang X, Wang M, Chen JW, Xiang JH, Xiang LJ, et al. Consistency, distinction and potential metabolic crosstalk of nitrogen mobilization-related genes in silk production and silk gland biology. Zool Res. 2025;46(2):446.
48. Zhang D, Hussain A, Manghwar H, Xie K, Xie S, Zhao S, et al. Genome editing with the CRISPR-Cas system: An art, ethics and global regulatory perspective. Plant Biotechnol J. 2020;18(8):1654-1675.
49. Zhang Q, Cheng T, Sun Y, Wang Y, Feng T, Li X, Liu L, et al. Synergism of open chromatin regions involved in regulating genes in Bombyx mori. Insect Biochem Mol Biol. 2019;110:10-18.
50. Zhang Q, Hua X, Sun Y, Lin Z, Cao Y, Zhao P, Xia Q. Dynamic chromatin conformation and accessibility changes mediate the spatial-specific gene regulatory network in Bombyx mori. Int J Biol Macromol. 2023;124415.
51. Zhang Y, Liu H, Li F, Chen K, Xiang Z. Identification of yield-related quantitative trait loci and candidate genes for cocoon traits in Bombyx mori using highdensity linkage maps. Sci Rep. 2018;8:11273.
52. Zhao P, Wang G, Yu Z, Xia Q. Functional analysis of BmVps13d reveals its essential role in silk gland development and silk yield in Bombyx mori. Dev Comp Immunol. 2019;97:103-110.
53. Zhou S, Gu Y, Dai H, Zhou X, Qin S, Li M, Sun X. The impact of the BmSur8 gene encoding a scaffold protein on cocoon silk yield in Bombyx mori. J Econ Entomol. 2025;118(4):1922-1932.