Establishment of CRISPR-Cas9 ribonucleoprotein mediated MSTN gene edited pregnancy in buffalo: Compare cells transfection and zygotes electroporation

IF 2.4 2区 农林科学 Q3 REPRODUCTIVE BIOLOGY
Meeti Punetha , Sheetal Saini , Suman Choudhary , Surabhi Sharma, Renu Bala, Pradeep Kumar, R.K. Sharma, P.S. Yadav, T.K. Datta, Dharmendra Kumar
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Abstract

Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance.

在水牛体内建立 CRISPR-Cas9 核糖核蛋白介导的 MSTN 基因编辑妊娠:比较细胞转染和胚胎电穿孔
基因组编辑被认为是农业和研究领域的一种强大工具,它能提高我们对遗传功能、疾病和生产力的认识。然而,由于妊娠期长、单胎妊娠和饲养成本高昂等诸多挑战,基因组编辑在水牛身上的进展一直落后于其他哺乳动物。在本研究中,我们的目标是产生MSTN编辑的水牛,作为概念验证。为了实现这一目标,我们采用了体细胞核移植(SCNT)和子代电穿孔(CRISPR-EP)技术。为此,我们首先确定了将 RNP 复合物导入成纤维细胞的最佳转染方法,并将其进一步用于 SCNT。为此,我们比较了成纤维细胞中核转染和脂质体转染的转染、裂解效率和细胞活力。结果发现,核染组的裂解率、转染效率和细胞存活率明显高于脂染组(P ≤ 0.05)。通过核转染产生了 4 个 MSTN 编辑菌落,其中 3 个菌落为双拷贝,1 个为单拷贝。此外,我们还比较了 SCNT 和子代电穿孔的效果、胚胎发育潜能和随后的妊娠结果。结果发现,电穿孔组的囊胚率明显高于 SCNT 组(P ≤ 0.05)。然而,子代电穿孔组有两例妊娠被证实是经 MSTN 编辑的。由于子代电穿孔不需要像 SCNT 那样复杂的显微操作技术,因此有可能促进水牛等大型家畜的基因改造。本研究为诱导具有实际或生物学意义的基因变异奠定了基础。
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来源期刊
Theriogenology
Theriogenology 农林科学-生殖生物学
CiteScore
5.50
自引率
14.30%
发文量
387
审稿时长
72 days
期刊介绍: Theriogenology provides an international forum for researchers, clinicians, and industry professionals in animal reproductive biology. This acclaimed journal publishes articles on a wide range of topics in reproductive and developmental biology, of domestic mammal, avian, and aquatic species as well as wild species which are the object of veterinary care in research or conservation programs.
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