小分子药物对CRISPR/Cas12i介导的两种绵羊原代细胞基因敲入效率的影响

doi:10.11843/j.issn.0366-6964.2025.12.015

Abstract

Abstract: Using primary ovine cells as materials, this study aimed to verify the gene editing efficiency of precise repair through homology-directed repair (HDR) with single-stranded oligodeoxynucleotide (ssODN) as the donor mediated by the CRISPR/Cas12i system, and to explore the effects of small molecule drugs on the efficiency of insertions and deletions (Indels) and the knock-in efficiency. In this study, two types of primary ovine cells were used as experimental subjects to verify the gene editing efficiency of the CRISPR/Cas12i system at 25 target gene loci. Then, 3 target sites (SOCS2-gRNA1, SOCS2-gRNA3, and TBXT-gRNA5) in ovine fetal fibroblasts and 5 target sites (MSTN-gRNA2, MSTN-gRNA5, Myf6-gRNA4, Myf6-gRNA5 and MyoG-gRNA4) in ovine hindlimb muscle cells were selected to detect the effects of 5 small molecule drugs (SCR7, Nocodazole, RS-1, Vorinostat, and Entinostat) on the Indel efficiency at each target site. Finally, the effect of the 5 small molecule drugs on the gene knock-in efficiency of precise repair through HDR with ssODN as the donor mediated by the CRISPR/Cas12i system was detected at the MSTN-gRNA2 target site. This study verified that the CRISPR/Cas12i system had high gene editing activity at 25 different target sites in the two types of primary ovine cells. After treatment with small molecule drugs, SCR7 decreased the Indels efficiency at each target site in primary ovine cells, while treatment with RS-1, Entinostat, and Vorinostat increased the Indels efficiency at each site. Nocodazole had inconsistent effects on the two types of cells. After treatment with the 5 small molecule drugs, the knock-in efficiency at the MSTN-gRNA2 site was increased to varying degrees. Among them, the treatment groups with 2 μmol·L^-1 Vorinostat and 4 μmol·L^-1 Entinostat had the highest enhancement effects, with the knock-in efficiencies being 7.67% and 7.73%, respectively. This experiment provides a reference for the application and promotion of the CRISPR/Cas12i gene editing system, offers new ideas for the creation of gene-edited sheep with excellent traits, and provides technical support for accelerating the work of precise molecular breeding.

Key words: CRISPR/Cas12i, homologous recombination, sheep, Indels, gene knock-in

CLC Number:

S826.2

SU Dawei, CHEN Qiuchong, MIAO Eryu, ZHANG Qijing, CHEN Zhe, WANG Xiaolong, XU Kun. The Effect of Small Molecule Drugs on Gene Knock-in Efficiency Mediated by the CRISPR/Cas12i System in Primary Ovine Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6104-6115.

References

[1] LIEBER M R. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway [J]. Annu Rev Biochem, 2010, 79: 181-211.
[2] CHANG H H Y, PANNUNZIO N R, ADACHI N, et al. Non-homologous DNA end joining and alternative pathways to double-strand break repair [J]. Nat Rev Mol Cell Biol, 2017, 18(8): 495-506.
[3] SUNG P, KLEIN H. Mechanism of homologous recombination: mediators and helicases take on regulatory functions [J]. Nat Rev Mol Cell Biol, 2006, 7(10): 739-750.
[4] YAN W X, HUNNEWELL P, ALFONSE L E, et al. Functionally diverse type V CRISPR-Cas systems [J]. Science, 2019, 363(6422): 88-91.
[5] HARRINGTON L B, MA E, CHEN J S, et al. A scoutRNA is required for some type V CRISPR-Cas systems [J]. Mol Cell, 2020, 79(3): 416-424.
e415.
[6] RAN F A, HSU P D, LIN C Y, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity [J]. Cell, 2013, 154(6): 1380-1389.
[7] MAKAROVA K S, WOLF Y I, IRANZO J, et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants [J]. Nat Rev Microbiol, 2020, 18(2): 67-83.
[8] ZETSCHE B, GOOTENBERG J S, ABUDAYYEH O O, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system [J]. Cell, 2015, 163(3): 759-771.
[9] GILLMORE J D, GANE E, TAUBEL J, et al. CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis [J]. N Engl J Med, 2021, 385(6): 493-502.
[10] 陈秋崇, 李尚朴, 苗洱钰, 等. HDACi和RS-1提高CRISPR/Cas12i介导的HDR编辑效率 [J]. 农业生物技术学报, 2024, 32(10): 2306-2323.
CHEN Q C, LI S P, MIAO E Y, et al. HDACi and RS-1 enhance CRISPR/Cas12i-mediated HDR editing efficiency [J]. Journal of Agricultural Biotechnology, 2024, 32(10): 2306-2323. (in Chinese)
[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 Genetics, 2015, 11(12): e1005629.
[12] INAGAKI-OHARA K, MAYUZUMI H, KATO S, et al. Enhancement of leptin receptor signaling by SOCS3 deficiency induces development of gastric tumors in mice [J]. Oncogene, 2014, 33(1): 74-84.
[13] LETELLIER E, HAAN S. SOCS2: physiological and pathological functions [J]. Front Biosci (Elite Ed), 2016, 8(1): 189-204.
[14] DOBIE R, MACRAE V E, PASS C, et al. Suppressor of cytokine signaling 2 (Socs2) deletion protects bone health of mice with DSS-induced inflammatory bowel disease [J]. Dis Model Mech, 2018, 11(1):dmm028456.
[15] BUCKINGHAM K J, MCMILLIN M J, BRASSIL M M, et al. Multiple mutant T alleles cause haploinsufficiency of Brachyury and short tails in Manx cats [J]. Mammalian Genome, 2013, 24(9): 400-408.
[16] KROMIK A, ULRICH R, KUSENDA M, et al. The mammalian cervical vertebrae blueprint depends on the T (brachyury) gene [J]. Genetics, 2015, 199(3): 873-883.
[17] JOULIA-EKAZA D, CABELLO G. The myostatin gene: physiology and pharmacological relevance [J]. Curr Opin Pharmacol, 2007, 7(3): 310-315.
[18] MCPHERRON A C, LAWLER A M, LEE S J. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member [J]. Nature, 1997, 387(6628): 83-90.
[19] WELLE S, BHATT K, PINKERT C A, et al. Muscle growth after postdevelopmental myostatin gene knockout [J]. Am J Physiol Endocrinol Metab, 2007, 292(4): E985-991.
[20] MEGENEY L A, RUDNICKI M A. Determination versus differentiation and the MyoD family of transcription factors [J]. Biochem Cell Biol, 1995, 73(9-10): 723-732.
[21] ZHANG Z, XU F, ZHANG Y, et al. Cloning and expression of MyoG gene from Hu sheep and identification of its myogenic specificity [J]. Mol Biol Rep, 2014, 41(2): 1003-1013.
[22] 刘宏祥, 徐文娟, 朱春红, 等. 鸭胚胎发育中后期胸肌发育阻滞的RNA-seq分析 [J]. 中国农业科学, 2018, 51(22): 4373-4386.
LIU H X, XU W J, ZHU C H, et al. RNA-seq analysis on development arrest of duck pectoralis muscle during semi-late embryonic period[J]. Scientia Agricultura Sinica, 2018, 51(22): 4373-4386. (in Chinese)
[23] AASE-REMEDIOS M E, COLL-LLADÓ C, FERRIER D E K. More than one-to-four via 2R: evidence of an independent amphioxus expansion and two-gene ancestral vertebrate state for MyoD-related myogenic regulatory factors (MRFs) [J]. Mol Biol Evol, 2020, 37(10): 2966-2982.
[24] 汤展毅, 严云勤, 高学军, 等. 牛myf6基因克隆及在成纤维细胞中的表达 [J]. 东北农业大学学报, 2010, 41(11): 77-82,后插72.
TANG Z Y, YAN Y Q, GAO X J, et al. Cloning of bovine myf6 gene and expression gene in fibroblasts [J]. Journal of Northeast Agricultural University, 2010, 41(11): 77-82, after insert 72. (in Chinese)
[25] AIELLO D, PATEL K, LASAGNA E. The myostatin gene: an overview of mechanisms of action and its relevance to livestock animals [J]. Anim Genet, 2018, 49(6): 505-519.
[26] MARUYAMA T, DOUGAN S K, TRUTTMANN M C, et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining [J]. Nat Biotechnol, 2015, 33(5): 538-542.
[27] JANG D E, LEE J Y, LEE J H, et al. Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency [J]. Exp Mol Med, 2018, 50(4): 1-9.
[28] RAHMANI B, KHEIRANDISH M H, GHANBARI S, et al. Targeting DNA repair pathways with B02 and Nocodazole small molecules to improve CRIS-PITCh mediated cassette integration in CHO-K1 cells [J]. Sci Rep, 2023, 13(1): 3116.
[29] SETO E, YOSHIDA M. Erasers of histone acetylation: the histone deacetylase enzymes [J]. Cold Spring Harb Perspect Biol, 2014, 6(4): a018713.
[30] ZHANG J P, YANG Z X, ZHANG F, et al. HDAC inhibitors improve CRISPR-mediated HDR editing efficiency in iPSCs [J]. Sci China Life Sci, 2021, 64(9): 1449-1462.
[31] 黄兰兰, 石国庆, 白洁. 绵羊胚胎成纤维细胞的体外培养 [J]. 黑龙江动物繁殖, 2006, (2): 4-5.
HUANG L L, SHI G Q, BAI J. In vitro culture of sheep embryonic fibroblasts [J]. Heilongjiang Journal of Animal Reproduction, 2006, (2): 4-5. (in Chinese)
[32] MCGAW C, GARRITY A J, MUNOZ G Z, et al. Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing [J]. Nat Commun, 2022, 13(1): 2833.
[33] 谢洪涛, 段志强. 利用Cas12i在大豆中进行基因编辑的方法, CN116218896A [P/OL]. XIE H T, DUAN Z Q. Method for gene editing in soybeans using Cas12i, CN116218896A [P/OL]. (in Chinese)
[34] 姚方瑶. 绵羊CRISPR/Cas12i基因编辑技术体系的优化与应用 [D]. 杨凌：西北农林科技大学, 2024.
YAO F Y. Optimisation and Application of the CRISPR/Cas12i Gene Editing Technology System for Sheep [D]. Yangling: Northwest A&F University, 2024.(in Chinese)
[35] MANJUNATH M, CHOUDHARY B, RAGHAVAN S C. SCR7, a potent cancer therapeutic agent and a biochemical inhibitor of nonhomologous DNA end-joining [J]. Cancer Rep (Hoboken), 2021, 4(3): e1341.
[36] VARTAK S V, RAGHAVAN S C. Inhibition of nonhomologous end joining to increase the specificity of CRISPR/Cas9 genome editing [J]. Febs J, 2015, 282(22): 4289-4294.
[37] LIU B, CHEN S, ROSE A, et al. Inhibition of histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing [J]. Nucleic Acids Res, 2020, 48(2): 517-532.
[38] JOGLEKAR A V, STEIN L, HO M, et al. Dissecting the mechanism of histone deacetylase inhibitors to enhance the activity of zinc finger nucleases delivered by integrase-defective lentiviral vectors [J]. Hum Gene Ther, 2014, 25(7): 599-608.
[39] YOON S, EOM G H. HDAC and HDAC inhibitor: From cancer to cardiovascular diseases [J]. Chonnam Med J, 2016, 52(1): 1-11.
[40] KUSCU C, ARSLAN S, SINGH R, et al. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease [J]. Nat Biotechnol, 2014, 32(7): 677-683.
[41] CHARI R, MALI P, MOOSBURNER M, et al. Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach [J]. Nat Methods, 2015, 12(9): 823-826.
[42] MORENO-MATEOS M A, VEJNAR C E, BEAUDOIN J D, et al. CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo [J]. Nat Methods, 2015, 12(10): 982-988.
[43] ORTHWEIN A, NOORDERMEER S M, WILSON M D, et al. RETRACTED ARTICLE: A mechanism for the suppression of homologous recombination in G1 cells [J]. Nature, 2015, 528(7582): 422-426.
[44] CHAPMAN J R, TAYLOR MARTIN R G, BOULTON SIMON J. Playing the end game: DNA double-strand break repair pathway choice [J]. Molecular Cell, 2012, 47(4): 497-510.
[45] RICHARDSON C D, RAY G J, DEWITT M A, et al. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA [J]. Nat Biotechnol, 2016, 34(3): 339-344.
[46] YANG L, GUELL M, BYRNE S, et al. Optimization of scarless human stem cell genome editing [J]. Nucleic Acids Res, 2013, 41(19): 9049-9061.

The Effect of Small Molecule Drugs on Gene Knock-in Efficiency Mediated by the CRISPR/Cas12i System in Primary Ovine Cells

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	BAI Feng, MAERZIYA·Yasen , AMINIGULI·Abulaizi , TENG Wen, LUO Chunyan, NAZHAKAITI·Ainiwaner , ZHANG Yuntao, JI Xinmin, ZHANG Yanhua. Genome-Wide Association Study of Body Weight and Body Size Traits In Turpan Black Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4315-4327.
[2]	XING Zhou, SONG Chenglei, CAO Fengfeng, LI Zhuoying, LI Qingyun, TAO Jinzhong*. Effects of Plasma Steroid Hormones on Superovulation Efficiency in Tan Sheep after Superovulation Treatment [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4422-4431.
[3]	LIU Can, SU Yixin, JING Xianjin, LI Wenze, YANG Lepu, WANG Ruijun, ZHANG Yanjun, WANG Zhiying, LÜ Qi, SU Rui. Research Progress of Epigenetics in Sheep and Goats Genetic Breeding [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 3561-3577.
[4]	WEI Kangkang, MA Gui, LI Wendi, TIAN Yu, ZHANG Lingkai, ZHU Jihong, HU Yamei. Research Progress of Single-Cell Sequencing Technology in the Growth and Development Process of Sheep Ovaries [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3080-3087.
[5]	ZHANG Jialiang, HUANG Chang, YANG Yonglin, YANG Hua, BAI Wenlin, MA Yuehui, ZHAO Qianjun. Genetic Structure and Wool Trait Selection Signatures Analysis of Chinese Sheep Populations Based on 50K Liquid SNP Chip [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3164-3176.
[6]	LAN Mingxi, QIN Qing, ZHANG Chongyan, LIU Zhichen, ZHANG Jingwen, ZHAO Dan, WU Danni, QIN Tian, WANG Zhixin, LIU Zhihong. Determination and Analysis of Slaughtering Performance and Meat Quality of Different Parts of Ujumqin Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3177-3187.
[7]	LUO Ruijie, WANG Jiankui, CAO Suying. Integrated Sequencing Analysis of lncRNA and mRNA Related to Ancestral-like Coarse in Aohan Fine Wool Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2685-2700.
[8]	ZHU Aiwen, WANG Jian, ZHU Gehui, LIU Haixia, PINGCUO Bandan, WANG Jun, DEQING Zhuoga, YAN Wei, HAN Dayong. Zearalenone Induced Proliferation, Apoptosis, Oxidative Stress and NAC Protective Mechanism of Sertoli Cells in Pengbo Semi-fine Wool Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2752-2764.
[9]	GU Bo, WANG Anqi, YU Xinmiao, GUO Juntong, YANG Yi, DENG Yijie, JIANG Huaizhi. Construction of Ovarian ceRNA Networks and Screening of Key miRNA in Two Different Breeds of Sheep Based on Whole Transcription Sequencing [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2765-2777.
[10]	QIAO Liying, WANG Wannian, ZHANG Li, PANG Zhixu, ZHANG Siying, LI Yifan, LIU Wenzhong. Machine Learning Methods for Sheep Breed Classification Based on Genomic Markers [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2157-2167.
[11]	SUN Guoxin, LI Yunhua, SAI Yin, GUO Wenhua, ZHAO Yanhong, ZHANG Manxin, LIU Jiasen. Population Structure Analysis and Economic Traits Related Selection Signal Detection of Hu Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2168-2181.
[12]	LI Xiaowei, TIAN Wei, LIU Yuan, LI Huixia. Study on the Difference of m⁶A Methylation Modification in Ovarian Granulosa Cells of Hu Sheep under Heat Stress [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1712-1721.
[13]	MA Yingtian, JIANG Luyao, LI Zengkai, QIN Jianping, ZHAO Jianhua, HE Yufang, SONG Yuxuan, ZHANG Lei. Effect of Cyanidin-3-rutinoside on Cryopreservation of Semen of Dairy Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1768-1778.
[14]	YANG Yang, LI Liangyuan, WAN Pengcheng, LU Shouliang, LIU Changbin, YANG Hua, WANG Limin, DAI Rong, ZHOU Ping. Screening and Analysis of Core Genes and Key lncRNAs for Seasonal Estrus Traits in Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1264-1277.
[15]	HE Yu, WANG Xiangyu, DI Ran, CHU Mingxing, LIANG Chen. BMP4/SMAD4 Downregulates GJA1 Gene Expression to Affect the Gap Junctional Intercellular Communication Activity in Sheep Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 679-688.