CRISPR/Cas9技术高效制备山羊SOCS2基因编辑胚胎

doi:10.11843/j.issn.0366-6964.2024.01.014

Abstract

Abstract: This study aimed to design and screen the sgRNAs for efficiently editing SOCS2 gene, a growth suppressor factor, in the parthenogenetic embryos of goats, laying a technical foundation for the creation of fast-growing goat. In this study, two sgRNAs were designed for the SH2 domain conserved sequence of goat SOCS2 gene by online website, and the corresponding expression vector was constructed. sgRNA and Cas9 mRNA were obtained by in vitro transcription. The target DNA cleavage efficiency of sgRNA+Cas9 protein was detected in vitro. On this basis, 442 parthenogenetic embryos isolated and cultured from goat ovaries in slaughterhouse were divided into 3 groups. Two experimental groups were microinjected with sgRNA and Cas9 mRNA mixture, and the control group was injected with ultra-pure water. Using the whole genome DNA amplification product of a single blastocyst as a template, the target region was amplified by PCR and sequenced, and the edited samples were sequenced to test editing varieties by T-A clone. According to the prediction of online websites, each sgRNA selected 5 potential off-target sites with the least number of mismatches for off-target detection. The results showed that two sgRNAs were obtained near amino acid codon 83 and 96 of SOCS2 gene, and their expression vectors were successfully constructed. High quality sgRNA and Cas9 mRNA were obtained by in vitro transcription. Both sgRNAs could guide the Cas9 protein to completely cleave the target DNA in vitro. The editing efficiency of SOCS2-sg-83 to embryos was 94.1%. Among 160 clones, 152 were inserted, deleted or replaced at the target site, with a probability of 95.0%, of which 81.3% had frameshift mutation, 9.4% of the clones destroyed the SH2 domain, and accumulated 90.6% of the clones achieved functional knockout of SOCS2 gene. The editing efficiency of SOCS2-sg-96 to embryos was 50.0%, among the 80 clones, 70 clones were inserted, deleted or replaced at the target site, with a probability of 87.5%, of which 75.0% clones had frameshift mutation, 5.0% of the clones lost amino acid codon 96, and accumulated 80.0% of the clones achieved functional knockout of the SOCS2 gene. In addition, no off target was found in all edited embryos. In conclusion, SOCS2-sg-83 can achieve efficient and accurate editing and knockout of SOCS2 gene in goat embryos, which laying a technical foundation for the efficient preparation of SOCS2 gene knockout goat and breeding of fast-growing mutton goat.

Key words: goat embryos, SOCS2, CRISPR/Cas9, gene editing

CLC Number:

S827.2

ZHANG Chenjian, LI Yinxia, DING Qiang, LIU Weijia, WANG Huili, HE Nan, WU Jiashun, CAO Shaoxian. Efficient Preparation of CRISPR/Cas9-mediated Goat SOCS2 Gene Edited Embryos[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 129-141.

References 42

[1]	GREENHALGH C J, RICO-BAUTISTA E, LORENTZON M, et al.SOCS2 negatively regulates growth hormone action in vitro and in vivo[J].J Clin Invest, 2005, 115(2):397-406.
[2]	VESTERLUND M, ZADJALI F, PERSSON T, et al.The SOCS2 ubiquitin ligase complex regulates growth hormone receptor levels[J].PLoS One, 2011, 6(9):e25358.
[3]	李亭亭, 刘秋月, 李向臣, 等.绵羊经济性状相关基因研究进展及其应用[J].畜牧兽医学报, 2022, 53(8):2417-2434.LI T T, LIU Q Y, LI X C, et al.Research progress and applications of genes associated with economic traits in sheep[J].Acta Veterinaria et Zootechnica Sinica, 2022, 53(8):2417-2434.(in Chinese)
[4]	METCALF D, GREENHALGH C J, VINEY E, et al.Gigantism in mice lacking suppressor of cytokine signalling-2[J].Nature, 2000, 405(6790):1069-1073.
[5]	STARR R, WILLSON T A, VINEY E M, et al.A family of cytokine-inducible inhibitors of signalling[J].Nature, 1997, 387(6636):917-921.
[6]	LI K L, MEZA GUZMAN L G, WHITEHEAD L, et al.SOCS2 regulation of growth hormone signaling requires a canonical interaction with phosphotyrosine[J].Biosci Rep, 2022, 42(12):BSR20221683.
[7]	BULLOCK A N, DEBRECZENI J É, EDWARDS A M, et al.Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase[J].Proc Natl Acad Sci U S A, 2006, 103(20):7637-7642.
[8]	BULATOV E, MARTIN E M, CHATTERJEE S, et al.Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase:suppressor of cytokine signaling 2 (SOCS2)-elongin BC-cullin 5-ring box protein 2 (RBX2)[J].J Biol Chem, 2015, 290(7):4178-4191.
[9]	KUNG W W, RAMACHANDRAN S, MAKUKHIN N, et al.Structural insights into substrate recognition by the SOCS2 E3 ubiquitin ligase[J].Nat Commun, 2019, 10(1):2534.
[10]	LINOSSI E M, LI K L, VEGGIANI G, et al.Discovery of an exosite on the SOCS2-SH2 domain that enhances SH2 binding to phosphorylated ligands[J].Nat Commun, 2021, 12(1):7032.
[11]	RUPP R, SENIN P, SARRY J, et al.A point mutation in suppressor of cytokine signalling 2 (Socs2) increases the susceptibility to inflammation of the mammary gland while associated with higher body weight and size and higher milk production in a sheep model[J].PLoS Genet, 2015, 11(12):e1005629.
[12]	LIU Y, COTTLE W T, HA T.Mapping cellular responses to DNA double-strand breaks using CRISPR technologies[J].Trends Genet, 2023, 39(7):560-574.
[13]	MATHEW S M.Strategies for generation of mice via CRISPR/HDR-mediated knock-in[J].Mol Biol Rep, 2023, 50(4):3189-3204.
[14]	BU W, CREIGHTON C J, HEAVENER K S, et al.Efficient cancer modeling through CRISPR-Cas9/HDR-based somatic precision gene editing in mice[J].Sci Adv, 2023, 9(19):eade0059.
[15]	WANG Y, QI T, LIU J T, et al.A highly specific CRISPR-Cas12j nuclease enables allele-specific genome editing[J].Sci Adv, 2023, 9(6):eabo6405.
[16]	CONG L, RAN F A, COX D, et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science, 2013, 339(6121):819-823.
[17]	季海艳, 朱焕章.基因编辑技术在基因治疗中的应用进展[J].生命科学, 2015, 27(1):71-82.JI H Y, ZHU H Z.Progress of genome editing approaches towards gene therapy[J].Chinese Bulletin of Life Sciences, 2015, 27(1):71-82.(in Chinese)
[18]	殷文晶, 陈振概, 黄佳慧, 等.基于CRISPR-Cas9基因编辑技术在作物中的应用[J].生物工程学报, 2023, 39(2):399-424.YIN W J, CHEN Z G, HUANG J H, et al.Application of CRISPR-Cas9 gene editing technology in crop breeding[J].Chinese Journal of Biotechnology, 2023, 39(2):399-424.(in Chinese)
[19]	费晓钰, 石超群, 刘雪明, 等.CRISPR/Cas9系统介导的猪MRC1修饰基因降低PCV2复制的研究[J].畜牧兽医学报, 2023, 54(3):934-946.FEI X Y, SHI C Q, LIU X M, et al.CRISPR/Cas9 system mediated gene modificated MRC1 in PK15 cells reduce PCV2 replication[J].Acta Veterinaria et Zootechnica Sinica, 2023, 54(3):934-946.(in Chinese)
[20]	GUO C T, MA X T, GAO F, et al.Off-target effects in CRISPR/Cas9 gene editing[J].Front Bioeng Biotechnol, 2023, 11:1143157.
[21]	YANG Q Q, WU L L, MENG J, et al.EpiCas-DL:predicting sgRNA activity for CRISPR-mediated epigenome editing by deep learning[J].Comput Struct Biotechnol J, 2023, 21:202-211.
[22]	ZHOU S W, CAI B, HE C, et al.Programmable base editing of the sheep genome revealed no genome-wide off-target mutations[J].Front Genet, 2019, 10:215.
[23]	CHEN W, MCKENNA A, SCHREIBER J, et al.Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair[J].Nucleic Acids Res, 2019, 47(15):7989-8003.
[24]	CRISPO M, VILARIÑO M, DOS SANTOS-NETO P C, et al.Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis[J].Transgenic Res, 2015, 24(1):31-41.
[25]	LEDDA S, IDDA A, KELLY J, et al.A novel technique for in vitro maturation of sheep oocytes in a liquid marble microbioreactor[J]. J Assist Reprod Genet, 2016, 33(4):513-518.
[26]	UYTTENDAELE I, LEMMENS I, VERHEE A, et al.Mammalian protein-protein interaction trap (MAPPIT) analysis of STAT5, CIS, and SOCS2 interactions with the growth hormone receptor[J].Mol Endocrinol, 2007, 21(11):2821-2831.
[27]	尹智, 袁雪薇, 吕嘉伟, 等.CRISPR/Cas9系统介导的一步法胚胎注射获得猪GGTA1敲除胚胎[J].畜牧与兽医, 2016, 48(4):15-18.YIN Z, YUAN X W, LV J W, et al.One-step generation of GGTA1-knockout pig embryos by injection of CRISPR/Cas9 system[J].Animal Husbandry & Veterinary Medicine, 2016, 48(4):15-18.(in Chinese)
[28]	尉翔栋, 吕晨晨, 朱肖亭, 等.利用CRISPR-Cas9基因编辑技术制备牛MSTN基因编辑胚胎[J].河南农业科学, 2019, 48(2):131-136.YU X D, LV C C, ZHU X T, et al.Preparation of bovine MSTN gene edited embryos using CRISPR-Cas9 gene editing technology[J]. Journal of Henan Agricultural Sciences, 2019, 48(2):131-136.(in Chinese)
[29]	ZHANG X M, LI W R, WU Y S, et al.Disruption of the sheep BMPR-IB gene by CRISPR/Cas9 in in vitro-produced embryos[J].Theriogenology, 2017, 91:163-172.e2.
[30]	HUSZÁR K, WELKER Z, GYÖRGYPÁL Z, et al.Position-dependent sequence motif preferences of SpCas9 are largely determined by scaffold-complementary spacer motifs[J].Nucleic Acids Res, 2023, 51(11):5847-5863.
[31]	NOSHAY J M, WALKER T, ALEXANDER W G, et al.Quantum biological insights into CRISPR-Cas9 sgRNA efficiency from explainable-AI driven feature engineering[J].Nucleic Acids Res, 2023, doi:10.1093/nar/gkad736.
[32]	LIU Y, FAN R, YI J K, et al.A fusion framework of deep learning and machine learning for predicting sgRNA cleavage efficiency[J].Comput Biol Med, 2023, 165:107476.
[33]	DYKE E, BIJNAGTE-SCHOENMAKER C, WU K M, et al.Generation of induced pluripotent stem cell line carrying frameshift variants in NPHP1 (UCSFi001-A-68) using CRISPR/Cas9[J].Stem Cell Res, 2023, 68:103053.
[34]	ABEUOVA L, KALI B, TUSSIPKAN D, et al.CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato[J].Transgenic Res, 2023, doi:10.1007/s11248-023-00356-8.
[35]	JEONG Y J, CHO J, KWAK J, et al.Immortalization of primary marmoset skin fibroblasts by CRISPR-Cas9-mediated gene targeting[J].Anim Cells Syst (Seoul), 2022, 26(6):266-274.
[36]	QIU Z W, LIU M Z, CHEN Z H, et al.High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases[J].Nucleic Acids Res, 2013, 41(11):e120.
[37]	HU R, FAN Z Y, WANG B Y, et al.RAPID COMMUNICATION:generation of FGF5 knockout sheep via the CRISPR/Cas9 system[J].J Anim Sci, 2017, 95(5):2019-2024.
[38]	XU C L, RUAN M Z, RAGI S D, et al.CRISPR off-target analysis platforms[J].Methods Mol Biol, 2023, 2560:279-285.
[39]	TIAN R, CAO C, HE D, et al.Massively parallel CRISPR off-target detection enables rapid off-target prediction model building[J].Med, 2023, 4(7):478-492.e6.
[40]	WIENERT B, CROMER M K.CRISPR nuclease off-target activity and mitigation strategies[J].Front Genome Ed, 2022, 4:1050507.
[41]	郭日红.Cas9技术介导兔和山羊MSTN与CLPG1基因编辑的研究[D].南京:南京农业大学, 2017.GUO R H.Genome editing at MSTN and CLPG1 locus of rabbit and goat using Cas9[D].Nanjing:Nanjing Agricultural University, 2017.(in Chinese)
[42]	赵为民, 王慧利, 曹少先, 等.猪CD163基因的单碱基编辑研究[J].畜牧兽医学报, 2022, 53(4):1041-1050.ZHAO W M, WANG H L, CAO S X, et al.The study of base editing of porcine CD163 gene[J].Acta Veterinaria et Zootechnica Sinica, 2022, 53(4):1041-1050.(in Chinese)

Efficient Preparation of CRISPR/Cas9-mediated Goat SOCS2 Gene Edited Embryos

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