Generation of TRPS1 knockout mice by CRISPR/Cas9-mediated gene targeting

Abstract

Abstract: Objectives: This study aimed to construct and identify heterozygous mice knocked out of TRPS1 gene based on CRISPR/Cas9 technology. Methods: After self-mating of C57BL/6N mice, TRPS1 knockout mice were constructed by injecting fertilized eggs with Cas9/sgRNA, and the mouse genotypes of heritable mice were detected by tail. TRPS1 heterozygous knockout mice were mated with wild-type mice to obtain mice with stable genotypes. Results: In this experiment, the fertilized eggs were injected with cas9 / sgRNA, all breeding mice were identified by tail genotype, 18 TRPS1 knockout heterozygous mice were successfully constructed. Conclusion: In this study, we successfully constructed TRPS1 knockout heterozygous mice based on CRISPR / cas9 technology, which provided a research platform for further research on the role of TRPS1 in the occurrence, development and possible liver metastasis of colorectal cancer at the animal level.

Key words: CRISPR/Cas9, TRPS1, Colorectal cancer, Gene knockout mouse

Li Tengyan, Liu Wenjie, Zhao Hong, Cai Jianqiang. Generation of TRPS1 knockout mice by CRISPR/Cas9-mediated gene targeting[J]. Electronic Journal of Liver Tumor, 2020, 7(4): 38-43.

References

[1] CHEN W, ZHENG R, BAADE P D, et al.Cancer statistics in China, 2015[J]. CA Cancer J Clin, 2016, 66(2):115-132.
[2] ADAM R, DE GRAMONT A, FIGUERAS J, et al.Managing synchronous liver metastases from colorectal cancer: a multidisciplinary international consensus[J]. Cancer Treat Rev, 2015, 41(9):729-741.
[3] ENGSTRAND J, NILSSON H, STROMBERG C, et al.Colorectal cancer liver metastases - a population-based study on incidence, management and survival[J]. BMC Cancer, 2018, 18(1):78.
[4] VEEN T, SOREIDE K.Can molecular biomarkers replace a clinical risk score for resectable colorectal liver metastasis?[J]. World J Gastrointest Oncol, 2017, 9(3):98-104.
[5] RATTI F, FIORENTINI G, CIPRIANI F, et al.Laparoscopic vs Open Surgery for Colorectal Liver Metastases[J]. JAMA Surg, 2018, 153(11):1028-1035.
[6] HUANG J Z, CHEN M, ZENG M, et al.Down-regulation of TRPS1 stimulates epithelial-mesenchymal transition and metastasis through repression of FOXA1[J]. J Pathol, 2016, 239(2):186-196.
[7] NAKAMURA M, SUGITA K, TOKURA Y.A novel missense mutation in the TRPS1 gene in a case of trichorhinophalangeal syndrome type I (TRPS1) with fish-like malodour[J]. J Eur Acad Dermatol Venereol, 2010, 24(3):358-359.
[8] MAAS S M, SHAW A C, BIKKER H, et al.Phenotype and genotype in 103 patients with tricho-rhino-phalangeal syndrome[J]. Eur J Med Genet, 2015, 58(5):279-292.
[9] ELSTER D, TOLLOT M, SCHLEGELMILCH K, et al.TRPS1 shapes YAP/TEAD-dependent transcription in breast cancer cells[J]. Nat Commun, 2018, 9(1):3115.
[10] WITWICKI R M, EKRAM M B, QIU X, et al.TRPS1 Is a Lineage-Specific Transcriptional Dependency in Breast Cancer[J]. Cell Reports, 2018, 25(5):1255-1267.
[11] SERANDOUR A A, MOHAMMED H, MIREMADI A, et al.TRPS1 regulates oestrogen receptor binding and histone acetylation at enhancers[J]. Oncogene, 2018, 37(39):5281-5291.
[12] HONG J, SUN J, HUANG T.Increased expression of TRPS1 affects tumor progression and correlates with patients' prognosis of colon cancer[J]. Biomed Res Int, 2013, 2013:454085.
[13] HOU J, PARRISH J, LUDECKE H J, et al.A 4-megabase YAC contig that spans the Langer-Giedion syndrome region on human chromosome 8q24.1: use in refining the location of the trichorhinophalangeal syndrome and multiple exostoses genes (TRPS1 and EXT1)[J]. Genomics, 1995, 29(1):87-97.
[14] KAISER F J, LUDECKE H J, WEGER S.SUMOylation modulates transcriptional repression by TRPS1[J]. Biol Chem, 2007, 388(4):381-390.
[15] KARACA A, REYES M, SHUMATE L T, et al.Severe brachydactyly and short stature resulting from a novel pathogenic TRPS1 variant within the GATA DNA-binding domain[J]. Bone, 2019, 123:153-158.
[16] MALIK T H, Von STECHOW D, BRONSON R T, et al.Deletion of the GATA domain of TRPS1 causes an absence of facial hair and provides new insights into the bone disorder in inherited tricho-rhino-phalangeal syndromes[J]. Mol Cell Biol, 2002, 22(24):8592-8600.
[17] GOSS M, SOCORRO M, MONIER D, et al.Trps1 transcription factor regulates mineralization of dental tissues and proliferation of tooth organ cells[J]. Mol Genet Metab, 2019, 126(4):504-512.
[18] ADLI M.The CRISPR tool kit for genome editing and beyond[J]. Nat Commun, 2018, 9(1):1911.
[19] MIRZA Z, KARIM S.Advancements in CRISPR/Cas9 technology-Focusing on cancer therapeutics and beyond[J]. Semin Cell Dev Biol, 2019, 96:13-21.
[20] TORRES-RUIZ R, RODRIGUEZ-PERALES S.CRISPR-Cas9: A Revolutionary Tool for Cancer Modelling[J]. Int J Mol Sci, 2015, 16(9):22151-22168.
[21] REPLOGLE J M, NORMAN T M, XU A, et al.Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing[J]. Nat Biotechnol, 2020, 38(8):954-961.
[22] KOO T, LEE J, KIM J.Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9[J]. Molecules and Cells, 2015, 38(6):475-481.
[23] ZISCHEWSKI J, FISCHER R, BORTESI L.Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases[J]. Biotechnol Adv, 2017, 35(1):95-104.
[24] LIN J, WONG K C.Off-target predictions in CRISPR-Cas9 gene editing using deep learning[J]. Bioinformatics, 2018, 34(17):i656-i663.
[25] JIANG F, DOUDNA J A.CRISPR-Cas9 Structures and Mechanisms[J]. Annu Rev Biophys, 2017, 46:505-529.
[26] XU M, XU H, CHEN J, et al.Generation of conditional Acvrl1 knockout mice by CRISPR/Cas9-mediated gene targeting[J]. Mol Cell Probes, 2018, 37:32-38.