JIA-2021-1394 I型胶原α1链基因是猪基因组中的一个可选的友好基因座

doi:10.1016/j.jia.2022.08.105

Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (1): 202-213.DOI: 10.1016/j.jia.2022.08.105

JIA-2021-1394 I型胶原α1链基因是猪基因组中的一个可选的友好基因座

收稿日期:2021-08-11 接受日期:2021-12-20 出版日期:2023-01-20 发布日期:2021-12-20

The collagen type I alpha 1 chain gene is an alternative safe harbor locus in the porcine genome

XIANG Guang-ming^1*, ZHANG Xiu-ling^1*, XU Chang-jiang¹, FAN Zi-yao¹, XU Kui¹, WANG Nan¹, WANG Yue^{1, 2}, CHE Jing-jing¹, XU Song-song^{1, 3}, MU Yu-lian¹, LI Kui^{1, 3}, LIU Zhi-guo¹

¹State Key Laboratory of Animal Nutrition/Key Laboratory of Animal Genetics Breeding and Reproduction of Ministry of Agriculture and Rural Affairs of China, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China
² School of Life Science and Engineering, Foshan University, Foshan 528231, P.R.China
³ Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, P.R.China

Received:2021-08-11 Accepted:2021-12-20 Online:2023-01-20 Published:2021-12-20
About author:Received 11 August, 2021 Accepted 20 December, 2021 XIANG Guang-ming, E-mail: guangming202012@163.com; ZHANG Xiu-ling, E-mail: xiulingzhang2014@163.com; Correspondence LIU Zhi-guo, E-mail: zhiguoliu2010@126.com * These authors contributed equally to this study.
Supported by:
This work was financially supported by the Major Scientific Research Tasks for Scientific and Technological Innovation Projects of the Chinese Academy of Agricultural Sciences (CAAS-ZDRW202006), the National Transgenic Breeding Project (2018ZX08010-10B) and the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (ASTIP-IAS05).

摘要/Abstract

摘要：

外源基因在转基因动物和细胞中稳定、高效的表达，对于基因功能的研究和生物反应器的建立至关重要。动物基因组中的友好基因座能使外源基因高效稳定的表达且无副作用，但目前猪基因组中可供外源基因安全、高效定点整合的基因座相对较少，限制了多转基因猪的研究和发展。本研究提出了一种将猪I型胶原α1链（collagen type I alpha 1 chain，COL1A1）基因座作为友好基因座表达外源基因的新策略。利用CRISPOR软件设计了一对靶向COL1A1基因终止密码子的sgRNA，并连接到CRISPR/Cas9表达载体pX330中；同时合成了一个不含启动子、左右同源臂各长为900 bp的2A-GFP供体载体，然后共转染猪肾上皮细胞（porcine kidney epithelial，PK15)，胎儿成纤维细胞（porcine embryonic fibroblast，PEF）以及回肠上皮细胞（porcine intestinal epithelial，IPI-2I）3种细胞。电转染48 h后观察3种细胞的荧光情况，72 h后通过流式细胞术分选绿色荧光蛋白阳性的细胞，并通过荧光定量、蛋白质印记、转录组测序和CCK8实验在不同水平上评估COL1A1基因作为猪基因组外源基因定点整合位点的安全性。结果表明，共转染sgRNA和供体载体之后，可以分别在猪3种不同细胞中观察到绿色荧光，说明同源重组介导的定点整合系统可以在猪基因组中很好的发挥作用。qPCR结果显示，GFP敲入对3种细胞COL1A1基因mRNA的表达并无显著影响（PK15、PEF和IPI-2I细胞中的P值分别为0.29、0.66和0.20）。同样，蛋白质印记结果显示，GFP阳性细胞与野生型细胞COL1A1蛋白的表达并无显著差异（PK15、PEF和IPI-2I细胞中的P值分别为0.64、0.48和0.80)。转录组测序结果显示，GFP阳性PEF细胞与野生型PEF细胞的转录组显著正相关（P<2.2e-16），表明GFP敲入没有改变内源性基因的整体表达。CCK8实验表明，GFP敲入对PK15细胞增殖并无显著影响（24 h，48 h，72 h，96 h，120 h的P值分别为0.31，0.96，0.24，0.17和0.38）。上述结果表明，COL1A1基因可作为猪基因组中外源基因定点整合的友好基因座，可广泛应用于家畜育种和生物医学模型的建立。本研究首次鉴定出了COL1A1基因可以作为猪基因组中的友好基因座。

Abstract:

Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors. Safe harbor loci in the animal genome enable consistent overexpression of foreign genes, without side effects. However, relatively few safe harbor loci are available in pigs, a fact which has impeded the development of multi-transgenic pig research. We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain (COL1A1) gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. After the knock-in of a 2A peptide-green fluorescence protein (2A-GFP) transgene in the last codon of COL1A1 in multiple porcine cells, including porcine kidney epithelial (PK15), porcine embryonic fibroblast (PEF) and porcine intestinal epithelial (IPI-2I) cells, quantitative PCR (qPCR), Western blotting, RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus. The qPCR results showed that the GFP knock-in had no effect (P=0.29, P=0.66 and P=0.20 for PK15, PEF and IPI-2I cells, respectively) on the mRNA expression of COL1A1 gene. Similarly, no significant differences (P=0.64, P=0.48 and P=0.80 for PK15, PEF and IPI-2I cells, respectively) were found between the GFP knock-in and wild type cells by Western blotting. RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation (P<2.2e–16) with that of the wild type cells, indicating that the GFP knock-in did not alter the global expression of endogenous genes. Furthermore, the CCK8 assay showed that the GFP knock-in events had no adverse effects (P_24h=0.31, P_48h=0.96, P_72h=0.24, P_96h=0.17, and P_120h=0.38) on cell proliferation of PK15 cells. These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment

Key words: COL1A1 gene , safe harbor , knock-in , CRISPR/Cas9 , pig

. JIA-2021-1394 I型胶原α1链基因是猪基因组中的一个可选的友好基因座[J]. Journal of Integrative Agriculture, 2023, 22(1): 202-213.

XIANG Guang-ming, ZHANG Xiu-ling, XU Chang-jiang, FAN Zi-yao, XU Kui, WANG Nan, WANG Yue, CHE Jing-jing, XU Song-song, MU Yu-lian, LI Kui, LIU Zhi-guo. The collagen type I alpha 1 chain gene is an alternative safe harbor locus in the porcine genome[J]. Journal of Integrative Agriculture, 2023, 22(1): 202-213.

参考文献

Beard C, Hochedlinger K, Plath K, Wutz A, Jaenisch R. 2006. Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis, 44, 23–28.
Bou-Gharios G, Ponticos M, Rajkumar V, Abraham D. 2004. Extra-cellular matrix in vascular networks. Cell Proliferation, 37, 207–220.
Capecchi M R. 2005. Gene targeting in mice: Functional analysis of the mammalian genome for the twenty-first century. Nature Reviews Genetics, 6, 507–512.
Concordet J P, Haeussler M. 2018. CRISPOR: Intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Research, 46, W242–W245.
ENCODE Project Consortium. 2012. An integrated encyclopedia of DNA elements in the human genome. Nature, 489, 57–74.
Han X S, Xiong Y C, Zhao C Z, Xie S S, Li C C, Li X Y, Liu X D, Li K, Zhao S H, Ruan J X. 2019. Identification of glyceraldehyde-3-phosphate dehydrogenase gene as an alternative safe harbor locus in pig genome. Genes (Basel), 10, 660–670.
Huang J, Wang A T, Huang C, Sun Y F, Song B X, Zhou R, Li L. 2020. Generation of marker-free pbd-2 knock-in pigs using the CRISPR/Cas9 and Cre/loxP systems. Genes (Basel), 11, 951.
Jia R, Wang C. 2020. MiR-29b-3p reverses cisplatin resistance by targeting COL1A1 in non-small-cell lung cancer A549/DDP cells. Cancer Management Research, 12, 2559–2566.
Karsenty G, Park R W. 1995. Regulation of type I collagen genes expression. International Reviews of Immunology, 12, 177–185.
Lai L X, Kolber-Simonds D, Park K W, Cheong H T, Greenstein J L, Im G S, Samuel M, Bonk A, Rieke A, Day B N, Murphy C N, Carter D B, Hawley R J, Prather R S. 2002. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science, 295, 1089–1092.
Li G L, Zhang X W, Wang H Q, Mo J X, Zhong C L, Shi J S, Zhou R, Li Z C, Yang H Q, Wu Z F, Liu D W. 2020. CRISPR/Cas9-mediated integration of large transgene into pig CEP112 locus. G3 - Genes Genomes Genetics, 10, 467–473.
Li X P, Yang Y, Bu L, Guo X G, Tang C C, Song J, Fan N N, Zhao B T, Ouyang Z, Liu Z M, Zhao Y, Yi X L, Quan L Q, Liu S C, Yang Z G, Ouyang H S, Chen Y E, Wang Z, Lai L X. 2014. Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing. Cell Research, 24, 501–504.
Ma L Y, Wang Y Z, Wang H T, Hu Y Q, Chen J Y, Tan T, Hu M, Liu X J, Zhang R, Xing Y M, Zhao Y Q, Hu X X, Li N. 2018. Screen and verification for transgene integration sites in pigs. Scientific Reports, 8, 7433–7443.
Mccreath K J, Howcroft J, Campbell K H, Colman A, Schnieke A E, Kind A J. 2000. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature, 405, 1066–1069.
Ohtsuka M. 2014. Development of pronuclear injection-based targeted transgenesis in mice through Cre-loxP site-specific recombination. Methods in Molecular Biology, 1194, 3–19.
Perleberg C, Kind A, Schnieke A. 2018. Genetically engineered pigs as models for human disease. Disease Models & Mechanisms, 11, 11–22.
Ranawakage D C, Okada K, Sugio K, Kawaguchi Y, Kuninobu-Bonkohara Y, Takada T, Kamachi Y. 2021. Efficient CRISPR-Cas9-mediated knock-in of composite tags in zebrafish using long ssDNA as a donor. Frontiers in Cell and Developmental Biology, 8, 598634.
Ruan J X, Li H G, Xu K, Wu T W, Wei J L, Zhou R, Liu Z G, Mu Y L, Yang S L, Ouyang H S, Chen-Tsai R Y, Li K. 2015. Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs. Scientific Reports, 5, 14253–14262.
Sadelain M, Papapetrou E P, Bushman F D. 2012. Safe harbours for the integration of new DNA in the human genome. Nature Reviews Cancer, 12, 51–58.
Shin S K, Kaiser E E, West F D. 2020. Alcohol induced brain and liver damage: Advantages of a porcine alcohol use disorder model. Frontiers in Physiology, 11, 592950.
Shintani Y, Hollingsworth M A, Wheelock M J, Johnson K R. 2006. Collagen I promotes metastasis in pancreatic cancer by activating c-Jun NH2-terminal kinase 1 and up-regulating N-cadherin expression. Cancer Research, 66, 11745–11753.
Tran N T, Sommermann T, Graf R, Trombke J, Pempe J, Petsch K, Kuhn R, Rajewsky K, Chu V T. 2019. Efficient CRISPR/Cas9-mediated gene knockin in mouse hematopoietic stem and progenitor cells. Cell Reports, 28, 3510–3522.
Wakasa Y, Ozawa K, Takaiwa F. 2009. Higher-level accumulation of foreign gene products in transgenic rice seeds by the callus-specific selection system. Journal of Bioscience and Bioengineering, 107, 78–83.
Wang K K, Ouyang H S, Xie Z C, Yao C G, Guo N N, Li M J, Jiao H P, Pang D X. 2015. Efficient generation of myostatin mutations in pigs using the CRISPR/Cas9 system. Scientific Reports, 5, 16623–16633.
Wang K K, Tang X C, Xie Z C, Zou X D, Li M J, Yuan H M, Guo N N, Ouyang H S, Jiao H P, Pang D X. 2017. CRISPR/Cas9-mediated knockout of myostatin in Chinese indigenous Erhualian pigs. Transgenic Research, 26, 799–805.
Whitworth K M, Rowland R R R, Ewen C L, Trible B R, Kerrigan M A, Cino-Ozuna A G, Samuel M S, Lightner J E, Mclaren D G, Mileham A J, Wells K D, Prather R S. 2016. Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nature Biotechnology, 34, 20–22.
Xie Z C, Pang D X, Wang K K, Li M J, Guo N N, Yuan H M, Li J N, Zou X D, Jiao H P, Ouyang H S, Li Z J, Tang X C. 2017. Optimization of a CRISPR/Cas9-mediated knock-in strategy at the porcine Rosa26 locus in porcine foetal fibroblasts. Scientific Reports, 7, 3036–3047.
Xiong Y C, Han X S, Zhang J F, Zhao G X, Wang Z C, Zhuang R Z, Nie X W, Xie S S, Li C C, Li X Y, Liu X D, Li K, Zhao S H, Ruan J X. 2020. Identification of ACTB gene as a potential safe harbor locus in pig genome. Molecular Biotechnology, 62, 589–597.
Xu K, Zhou Y R, Mu Y L, Liu Z G, Hou S H, Xiong Y J, Fang L R, Ge C L, Wei Y H, Zhang X L, Xu C J, Che J J, Fan Z Y, Xiang G M, Guo J K, Shang H T, Li H, Xiao S B, Li J L, Li K. 2020. CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance. eLife, 9, e57132–e57155.
Yang D S, Wang C E, Zhao B T, Li W, Ouyang Z, Liu Z M, Yang H Q, Fan P, O’Neill A, Gu W W, Yi H, Li S H, Lai L X, Li X J. 2010. Expression of Huntington’s disease protein results in apoptotic neurons in the brains of cloned transgenic pigs. Human Molecular Genetics, 19, 3983–3994.
Yang H Q, Wu Z F. 2018. Genome editing of pigs for agriculture and biomedicine. Frontiers in Genetics, 9, 360–371.
Yao X, Wang X, Hu X D, Liu Z, Liu J L, Zhou H B, Shen X W, Wei Y, Huang Z J, Ying W Q, Wang Y, Nie Y H, Zhang C C, Li S L, Cheng L P, Wang Q F, Wu Y, Huang P Y, Sun Q, Shi L Y, et al. 2017. Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Research, 27, 801–814.
Zhou Z P, Yang L L, Cao H, Chen Z R, Zhang Y, Wen X Y, Hu J. 2019. In vitro validation of a CRISPR-mediated CFTR correction strategy for preclinical translation in pigs. Human Gene Therapy, 30, 1101–1116.

JIA-2021-1394 I型胶原α1链基因是猪基因组中的一个可选的友好基因座

The collagen type I alpha 1 chain gene is an alternative safe harbor locus in the porcine genome

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