한국발생생물학회지:발생과생식 (Development and Reproduction)

  • 제27권4호
  • /
  • Pages.205-211
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  • 2023
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  • 2465-9525(pISSN)
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  • 2465-9541(eISSN)
한국발생생물학회 (The Korean Society of Developmental Biology)
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Generation of ints14 Knockout Zebrafish using CRISPR/Cas9 for the Study of Development and Disease Mechanisms

  • Ji Hye Jung (Department of Genetic Resources, 75 National Marine Biodiversity Institute of Korea) ;
  • Sanghoon Jeon (Department of Genetic Resources, 75 National Marine Biodiversity Institute of Korea) ;
  • Heabin Kim (Department of Genetic Resources, 75 National Marine Biodiversity Institute of Korea) ;
  • Seung-Hyun Jung (Department of Genetic Resources, 75 National Marine Biodiversity Institute of Korea)
  • 투고 : 2023.08.15
  • 심사 : 2023.11.25
  • 발행 : 2023.12.31
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초록

INTS14/VWA9, a component of the integrator complex subunits, plays a pivotal role in regulating the fate of numerous nascent RNAs transcribed by RNA polymerase II, particularly in the biogenesis of small nuclear RNAs and enhancer RNAs. Despite its significance, a comprehensive mutation model for developmental research has been lacking. To address this gap, we aimed to investigate the expression patterns of INTS14 during zebrafish embryonic development. We generated ints14 mutant strains using the CRISPR/Cas9 system. We validated the gRNA activity by co-injecting Cas9 protein and a single guide RNA into fertilized zebrafish eggs, subsequently confirming the presence of a 6- or 9-bp deletion in the ints14 gene. In addition, we examined the two mutant alleles through PCR analysis, T7E1 assay, TA-cloning, and sequencing. For the first time, we used the CRISPR/Cas9 system to create a model in which some sequences of the ints14 gene were removed. This breakthrough opens new avenues for in-depth exploration of the role of ints14 in animal diseases. The mutant strains generated in this study can provide a valuable resource for further investigations into the specific consequences of ints14 gene deletion during zebrafish development. This research establishes a foundation for future studies exploring the molecular mechanisms underlying the functions of ints14, its interactions with other genes or proteins, and its broader implications for biological processes.

키워드

과제정보

This work was supported by the National Marine Biodiversity Institute Research Program (2023M00400).

참고문헌

  1. Ansai S, Kinoshita M (2014) Targeted mutagenesis using CRISPR/Cas system in medaka. Biol Open 3:362-371. https://doi.org/10.1242/bio.20148177
  2. Aoyama Y, Moriya N, Tanaka S, Taniguchi T, Hosokawa H, Maegawa S (2015) A novel method for rearing zebrafish by using freshwater rotifers (Brachionus calyciflorus). Zebrafish 12:288-295. https://doi.org/10.1089/zeb.2014.1032
  3. Baillat D, Hakimi MA, Naar AM, Shilatifard A, Cooch N, Shiekhattar R (2005) Integrator, a multiprotein mediator of small nuclear RNA processing, associates with the C-terminal repeat of RNA polymerase II. Cell 123:265-276. https://doi.org/10.1016/j.cell.2005.08.019
  4. Chen J, Ezzeddine N, Waltenspiel B, Albrecht TR, Warren WD, Marzluff WF, Wagner EJ (2012) An RNAi screen identifies additional members of the Drosophila integrator complex and a requirement for cyclin C/Cdk8 in snRNA 3'-end formation. RNA 18:2148-2156. https://doi.org/10.1261/rna.035725.112
  5. Jung SH, Kim HS, Ryu JH, Gwak JW, Bae YK, Kim CH, Yeo SY (2012) Her4-positive population in the tectum opticum is proliferating neural precursors in the adult zebrafish brain. Mol Cells 33:627-632. https://doi.org/10.1007/s10059-012-0091-5
  6. Krall M, Htun S, Schnur RE, Brooks AS, Baker L, Campomanes AA, Lamont RE, Gripp KW, Care 4 Rare Canada Consortium, Schneidman-Duhovny D, Micheil Innes A, Mancini GMS, Slavotinek AM (2019) Biallelic sequence variants in INTS1 in patients with developmental delays, cataracts, and craniofacial anomalies. Eur J Hum Genet 27:582-593. https://doi.org/10.1038/s41431-018-0298-9
  7. Mendoza-Figueroa MS, Tatomer DC, Wilusz JE (2020) The integrator complex in transcription and development. Trends Biochem Sci 45:923-934. https://doi.org/10.1016/j.tibs.2020.07.004
  8. Oegema R, Baillat D, Schot R, van Unen LM, Brooks A, Kia SK, Hoogeboom AJM, Xia Z, Li W, Cesaroni M, Lequin MH, Slegtenhorst M, Dobyns WB, de Coo IFM, Verheijen FW, Kremer A, van der Spek PJ (2017) Human mutations in integrator complex subunits link transcriptome integrity to brain development. PLoS Genet 13:e1006923.
  9. Peart N, Sataluri A, Baillat D, Wagner EJ (2013) Non-mRNA 3' end formation: How the other half lives. Wiley Interdiscip Rev RNA 4:491-506. https://doi.org/10.1002/wrna.1174
  10. Rienzo M, Casamassimi A (2016) Integrator complex and transcription regulation: Recent findings and pathophysiology. Biochim Biophys Acta BBA Gene Regul Mech 1859:1269-1280. https://doi.org/10.1016/j.bbagrm.2016.07.008
  11. Sabath K, Staubli ML, Marti S, Leitner A, Moes M, Jonas S (2020) INTS10-INTS13-INTS14 form a functional module of integrator that binds nucleic acids and the cleavage module. Nat Commun 11:3422.
  12. Welsh SA, Gardini A (2023) Genomic regulation of transcription and RNA processing by the multitasking integrator complex. Nat Rev Mol Cell Biol 24:204-220. https://doi.org/10.1038/s41580-022-00534-2