CRISPR/Cas9-mediated Site Specific Integration of Foreign Genes in Pig (Sus scrofa) PSP Locus
JIN Wei, DAI Min-Min, LI De-Juan, FAN Bao-Liang*
1 College of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, China; 2 Hebei Animal Husbandry and Veterinary Institute, Baoding 071000, China; 3 College of Veterinary Medicine, Hebei Agricultural University, Baoding 071001, China; 4 College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
Abstract：As a novel bioreactor, the salivary gland bioreactor specifically expresses various foreign proteins by using the regulatory region of salivary secretory proteins. The commonly used salivary secretory protein is parotid secretory protein (PSP).The aim of this study is to establish a method for site-specific integration of foreign genes into PSP gene using the CRISPR/Cas9 system and analyze the off-target effect of the selected gRNA on PSP gene. Firstly, sgRNA sequences were designed online according to PSP gene sequence and 5 sgRNA sequences were selected. Then, using sgRNA in vitro transcription kit and Cas9 in vitro digestion kit, the sgRNA sequence with high in vitro activity which targeting PSP gene were screened out. At the same time the co-expression vectors of pCas9-sgRNA for sgRNA and Cas9 and a targeting vector pMD19-5'arm-NeoR-3'arm carrying the expression structure of neomycin resistance gene (NeoR) were constructed. Finally, sgRNA and Cas9 co-expression vector and target vector were co-transfected into porcine kidney cells (PK-15 cells), screened with neomycin, and single cell clones were isolated. Positive cell clones which was knocked-in neomycin resistance gene (NeoR) were identified by PCR and sequencing, the off-target effects of every sgRNA were analyzd. The results of in vitro enzyme digestion showed that sgRNA1, sgRNA3, sgRNA4 and sgRNA5 had higher activity in vitro. Sequencing results indicated that the 4 sgRNAs were successfully targeted the forigen gene into the PSP gene of Sus scrofa. The knock-in efficiency of sgRNA1 and sgRNA5 was 22.7% (5/22) and 26.1% (6/23), with 1 potential off-target site had off-target effect; The efficiency of sgRNA3 and sgRNA4 was 50.0% (12/24) and 42.1% (8/19), with no off-target effect at the 5 potential off-target sites. In this study, the method for gene editing of pig PSP gene was successfully established, and screening for sgRNAs that efficiently guide foreign genes into the pig PSP gene provides basic data for further research on transgenic Sus scrofa.
靳伟, 代敏敏, 李德娟, 樊宝良. CRISPR/Cas9介导的外源基因在猪PSP位点的定点整合[J]. 农业生物技术学报, 2019, 27(9): 1569-1581.
JIN Wei, DAI Min-Min, LI De-Juan, FAN Bao-Liang. CRISPR/Cas9-mediated Site Specific Integration of Foreign Genes in Pig (Sus scrofa) PSP Locus. 农业生物技术学报, 2019, 27(9): 1569-1581.
1 程佳月. 2011. 非洲鸵鸟胫骨的形态学特征及硼对其生长发育的影响[D]. 博士学位论文,华中农业大学, 导师: 彭克美, 李奎, pp. 89-90. (Cheng J Y.2011. The research about the morphological characteristics of tibia and the influence of boron to the tibia development of african ostrich[D]. Thesis for Ph D., Huazhong Agricultural University, supervisor: Peng K M, Li K, pp. 89-90.) 2 金福强. 2010. 股骨头缺血性坏死脂类代谢异常的临床研究[D]. 硕士学位论文, 河北医科大学, 导师: 扈文海, pp. 16-21. (Jin F Q.2010. Clinical study on lipid metabolism of avascular necrosis of femoral head[D]. Thesis for M.S., Hebei Medical University, supervisor: Hu W H, pp. 16-21.) 3 贺俊平, 唐好文, 尉秀平. 2005. 活血化瘀中药对鸡实验性骨折愈合过程血清碱性磷酸酶及骨痂钙盐沉积的影响[J]. 山西农业大学学报(自然科学版), 25(2): 119-122. (He J P, Tang H W, Wei X P.2005. Effects of chinese herbal medicine on serum alp and calcium of callus in the process of fracture healing in chickens[J]. Journal of Shanxi Agricultural University (Nature Science Edition), 25(2): 119-122.) 4 厉婷, 崔燎. 2008. 脂质代谢紊乱和骨质疏松[J]. 中国骨质疏松杂志, 14(6): 428-432. (Li T, Cui L.2008. Lipid metaboism disorders and osteoporosis[J]. Chinese Journal of Osteoporosis, 14(6): 428-432.) 5 李鹏飞, 姜敏, 史超颖, 等. 2016. 规模化养殖场肉鸡腿病情况调查[J].畜牧与兽医, 48(6): 116-118. (Li P F, Jiang M, Shi Y C, et al.2016. An investigation of broiler leg disorders in large-scale farms[J]. Animal Husbandry and Veterinary Medicine, 48(6): 116-118.) 6 沈良诚, MichaelP. Morris.1993. 美国对肉鸡腿疾的全国性调查情况[J]. 国外畜牧学(猪与禽), 3(06): 42-45. (Shen L C, Morris M.1993. A National survey of broiler leg diseases in the United States[J]. Animal Science Abroad (Pigs and Poultry), 3(06): 42-45.) 7 武书庚, 杨兹明, 齐广海, 等. 2000. 禽的骨骼畸形[J]. 国外畜牧科技, 27(5): 47-52. (Wu S G, Yang Z M, Qi G H, et al.2000. Skeletal deformity of poultry[J]. Animal Science Abroad, 27(5): 47-52.) 8 Whitehead C. 李芹. 2010. 欧洲肉鸡生产中的骨骼问题及其解决措施[J].中国家禽, 32(12): 39-40. (Whitehead C, Li Q.2010. The bone problems and solutions in European broiler production[J]. China poultry, 32(12): 39-40.) 9 王石莹, 闫素梅. 2009. 碱性磷酸酶在动物骨骼代谢中的研究进展[J]. 饲料博览, (4): 14-17. (Wang S Y, Yan S M. 2009. Research progress of alkaline phosphatase in bone metabolism of animals[J]. Feed Review, (4): 14-17.) 10 张国梅, 黎翠亨, 严鸿生, 等. 1994. 如东县鸡腿病病因的调查研究[J]. 畜牧与兽医, 26(5): 215-218. (Zhang G M, Li C H, Yan H S, et al.1994. Investigation on etiology of chicken leg weakness in Rudong county[J]. Animal Husbandry and Veterinary Medicine, 26(5): 215-218.) 11 张劳. 1993. 雄性肉鸡跗关节间外翻―内翻畸形的相关因素剖析[J]. 北京农业大学学报, 2(1): 115-118. (Zhang L.1993. Study on the feactor causing valugus-varus deformity of the intertasal joint in broiler males[J]. Acta Agriculture University Pekinensis, 2(1): 115-118.) 12 Bradshaw R H, Kirkden R D, Broom D M.2002. A Review of the aetiology and pathology of leg weakness in broilers in relation to welfare[J]. Avian and Poultry Biology Reviews, 13: 45-103. 13 Cruickshank J J, Sim J S.1986. Morphometric and radiographic characteristics of tibial bone of broiler chickens with twisted leg disorders[J]. Avian Diseases, 30(4): 699-708. 14 Erken H Y, Ofluoglu O, Aktas M, et al.2012. Effect of pentoxifylline on histopathological changes in steroid-induced osteonecrosis of femoral head: Experimental study in chicken[J]. International Orthopaedics, 36(7): 1523-1528. 15 Julian R J.1984. Valgus-varus deformity of the intertarsal joint in broiler chickens[J]. The Canadian Veterinary Journal. La Revue Veterinaire Canadienne, 25(6): 254-258. 16 Gao Y, Du Z Q, Feng C G, et al.2010. Identification of quantitative trait loci for shank length and growth at different development stages in chicken[J]. Animal Genetics, 41(1): 101-104. 17 Kestin S C, Gregory I N, Knowles T G, et al.1992. Prevalence of leg weakness in broiler chickens and its relationship with genotype[J]. Veterinary Record, 131(9): 190-194. 18 Leterrier C, Nys Y.1992. Clinical and anatomical differences in varus and valgus deformities of chick limbssuggest different aetio-pathogenesis[J]. Avian Pathology, 21(3): 429-442. 19 Packialakshmi B, Liyanage R, Lay J O, et al.2016. Proteomic changes in the plasma of broiler chickens with femoral head necrosis[J]. Biomarker Insights, 11: BMI.S38291. 20 Zhou Z L, Deng Y F, Tao Q S, et al.2009. Effects of Gushukang, a Chinese herbal medicine, on bone characteristics and osteoporosis in laying hens[J]. Poultry Science, 88(11): 2342-2345.
