Abstract
Transgenic technology is now widely used in biomedical and agricultural fields. Transgenesis is commonly achieved through random integration which might cause some uncertain consequences. The site-specific integration could avoid this disadvantage. This study aimed to screen and validate the best safe harbor (SH) locus for efficient porcine transgenesis. First, the cells carrying the EGFP reporter construct at four different SH loci (ROSA26, AAVS1, H11 and COL1A1) were achieved through CRSIPR/Cas9-mediated HDR. At the COL1A1 and ROSA26 loci, a higher mRNA and protein expression of EGFP was detected, and it was correlated with a lower level of DNA methylation of the EGFP promoter, hEF1α. A decreased H3K27me3 modification of the hEF1α promoter at the COL1A1 locus was also detected. For the safety of transgenesis at different SH locus, we found that transgenesis could relatively alter the expression of the adjacent endogenous genes, but the influence was limited. We also did not observe any off-target cleavage for the selected sgRNAs of the COL1A1 and ROSA26 loci. In conclusion, the COL1A1 and ROSA26 were confirmed to be the best two SH loci with the COL1A1 being more competitive for porcine transgenesis. This work would greatly facilitate porcine genome engineering and transgenic pig production.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
Not applicable.
References
Ahuja MR (1997) Transgenes and genetic instability. United States Department of Agriculture Forest Service General Technical Report RM, pp 90–100
Bird AP, Wolffe AP (1999) Methylation-induced repression—belts, braces, and chromatin. Cell 99:451–454. https://doi.org/10.1016/s0092-8674(00)81532-9
Capecchi MR (1989) Altering the genome by homologous recombination. Science 244:1288–1292. https://doi.org/10.1126/science.2660260
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. https://doi.org/10.1126/science.1231143
Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34:184–191. https://doi.org/10.1038/nbt.3437
Fairbairn L, Kapetanovic R, Sester DP, Hume DA (2011) The mononuclear phagocyte system of the pig as a model for understanding human innate immunity and disease. J Leukoc Biol 89:855–871. https://doi.org/10.1189/jlb.1110607
Finnegan J, McElroy D (1994) Transgene inactivation: plants fight back! Nat Biotechnol 12:883–888. https://doi.org/10.1038/nbt0994-883
Friedrich G, Soriano P (1991) Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev 5:1513–1523. https://doi.org/10.1101/gad.5.9.1513
Hippenmeyer S, Youn YH, Moon HM, Miyamichi K, Zong H, Wynshaw-Boris A, Luo L (2010) Genetic mosaic dissection of Lis1 and Ndel1 in neuronal migration. Neuron 68:695–709. https://doi.org/10.1016/j.neuron.2010.09.027
Hsiau T, Maures T, Waite K, Yang J, Kelso R, Holden K, Stoner R (2018) Inference of CRISPR edits from Sanger trace data. BioRxiv 251082
Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832. https://doi.org/10.1038/nbt.2647
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. https://doi.org/10.1126/science.1225829
Kalla D, Kind A, Schnieke A (2020) Genetically engineered pigs to study cancer. Int J Mol Sci 21:488. https://doi.org/10.3390/ijms21020488
Kumaki Y, Oda M, Okano M (2008) QUMA: quantification tool for methylation analysis. Nucleic Acids Res 36:W170-175. https://doi.org/10.1093/nar/gkn294
Li LC, Dahiya R (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18:1427–1431. https://doi.org/10.1093/bioinformatics/18.11.1427
Li Y, Tollefsbol TO (2011) DNA methylation detection: bisulfite genomic sequencing analysis. Methods Mol Biol 791:11–21. https://doi.org/10.1007/978-1-61779-316-5_2
Li X, Burnight ER, Cooney AL, Malani N, Brady T, Sander JD, Staber J, Wheelan SJ, Joung JK, McCray PB Jr, Bushman FD, Sinn PL, Craig NL (2013) piggyBac transposase tools for genome engineering. Proc Natl Acad Sci U S A 110:E2279-2287. https://doi.org/10.1073/pnas.1305987110
Li C, Brant E, Budak H, Zhang B (2021) CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement. J Zhejiang Univ Sci B 22:253–284. https://doi.org/10.1631/jzus.B2100009
Ma L, Wang Y, Wang H, Hu Y, Chen J, Tan T, Hu M, Liu X, Zhang R, Xing Y, Zhao Y, Hu X, Li N (2018) Screen and verification for transgene integration sites in pigs. Sci Rep 8:7433. https://doi.org/10.1038/s41598-018-24481-1
McCreath KJ, Howcroft J, Campbell KH, Colman A, Schnieke AE, Kind AJ (2000) Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 405:1066–1069. https://doi.org/10.1038/35016604
Meurens F, Summerfield A, Nauwynck H, Saif L, Gerdts V (2012) The pig: a model for human infectious diseases. Trends Microbiol 20:50–57. https://doi.org/10.1016/j.tim.2011.11.002
Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272:263–267. https://doi.org/10.1126/science.272.5259.263
Niu D, Wei HJ, Lin L, George H, Wang T, Lee IH, Zhao HY, Wang Y, Kan Y, Shrock E, Lesha E, Wang G, Luo Y, Qing Y, Jiao D, Zhao H, Zhou X, Wang S, Wei H, Guell M, Church GM, Yang L (2017) Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science 357:1303–1307. https://doi.org/10.1126/science.aan4187
Niu D, Ma X, Yuan T, Niu Y, Xu Y, Sun Z, Ping Y, Li W, Zhang J, Wang T, Church GM (2021) Porcine genome engineering for xenotransplantation. Adv Drug Deliv Rev 168:229–245. https://doi.org/10.1016/j.addr.2020.04.001
Ouyang H, Han J, Huang Y (2021) Pig cloning using somatic cell nuclear transfer. Methods Mol Biol 2239:1–18. https://doi.org/10.1007/978-1-0716-1084-8_1
Renner S, Fehlings C, Herbach N, Hofmann A, von Waldthausen DC, Kessler B, Ulrichs K, Chodnevskaja I, Moskalenko V, Amselgruber W, Goke B, Pfeifer A, Wanke R, Wolf E (2010) Glucose intolerance and reduced proliferation of pancreatic beta-cells in transgenic pigs with impaired glucose-dependent insulinotropic polypeptide function. Diabetes 59:1228–1238. https://doi.org/10.2337/db09-0519
Ruan J, Li H, Xu K, Wu T, Wei J, Zhou R, Liu Z, Mu Y, Yang S, Ouyang H, Chen-Tsai RY, Li K (2015) Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs. Sci Rep 5:14253. https://doi.org/10.1038/srep14253
Shen L, Waterland RA (2007) Methods of DNA methylation analysis. Curr Opin Clin Nutr Metab Care 10:576–581. https://doi.org/10.1097/MCO.0b013e3282bf6f43
Symington LS, Gautier J (2011) Double-strand break end resection and repair pathway choice. Annu Rev Genet 45:247–271. https://doi.org/10.1146/annurev-genet-110410-132435
van Rensburg R, Beyer I, Yao XY, Wang H, Denisenko O, Li ZY, Russell DW, Miller DG, Gregory P, Holmes M, Bomsztyk K, Lieber A (2013) Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells. Gene Ther 20:201–214. https://doi.org/10.1038/gt.2012.25
Xie Z, Pang D, Wang K, Li M, Guo N, Yuan H, Li J, Zou X, Jiao H, Ouyang H, Li Z, Tang X (2017) Optimization of a CRISPR/Cas9-mediated knock-in strategy at the porcine Rosa26 locus in porcine foetal fibroblasts. Sci Rep 7:3036. https://doi.org/10.1038/s41598-017-02785-y
Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy M, Henckaerts E, Bonham K, Abbott GW, Linden RM, Field LJ, Keller GM (2008) Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453:524–528. https://doi.org/10.1038/nature06894
Yang L, Guell M, Niu D, George H, Lesha E, Grishin D, Aach J, Shrock E, Xu W, Poci J, Cortazio R, Wilkinson RA, Fishman JA, Church G (2015) Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science 350:1101–1104. https://doi.org/10.1126/science.aad1191
Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13:134. https://doi.org/10.1186/1471-2105-13-134
Zhang B (2021) CRISPR/Cas gene therapy. J Cell Physiol 236:2459–2481. https://doi.org/10.1002/jcp.30064
Zhang N, Feng B, Ma X, Sun K, Xu G, Zhou Y (2019) Dapagliflozin improves left ventricular remodeling and aorta sympathetic tone in a pig model of heart failure with preserved ejection fraction. Cardiovasc Diabetol 18:107. https://doi.org/10.1186/s12933-019-0914-1
Funding
This work was supported by the Key Project of Natural Science Foundation of Zhejiang Province (LZ22C010003), Scientific and technological innovation leading talents project of Zhejiang Provincial “High-level Talents Special Support Plan” (2021R52043), Zhejiang Science and Technology Major Program on Agriculture New Variety Breeding (2021C02068-4), the Fundamental Research Funds for the Provincial Universities of Zhejiang (2020YQ007) and Zhejiang A&F University Talent Initiative Project (Nos: 2019FR022, 2019FR052, 2021FR004 and 2020FR033).
Author information
Authors and Affiliations
Contributions
XM designed the study, performed the data analysis and drafted the manuscript; WJZ, LW, RC, ZYZ, CYH, PPT and ZXS conducted the study; TW and JFZ performed the data analysis; DN, XD and LL supervised the work.
Corresponding authors
Ethics declarations
Ethics approval
All the experiment involving animals in this study were conducted followed EU standards for the protection of animals used for scientific purposes and approved by Animal Ethics Committee of Zhejiang A&F University.
Competing interests
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Ma, X., Zeng, W., Wang, L. et al. Validation of reliable safe harbor locus for efficient porcine transgenesis. Funct Integr Genomics 22, 553–563 (2022). https://doi.org/10.1007/s10142-022-00859-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10142-022-00859-3