Knockout of myostatin (MSTN) gene using CRISPR/Cas9 technology in order to produce knockout Varamini sheep embryos

Bazgiri, Maryam; Fayazi, Jamal; Salehi, Mohammad; Jajarmi, Vahid

doi:10.22059/jap.2024.370186.623774

Document Type : Research Paper

Authors

¹ Department of Animal Science, Faculty of Animal and Food Science, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Iran. E-mail: m‌‌_bazgiri_1989@asnrukh.ac.ir

² Corresponding Author, Department of Animal Science, Faculty of Animal and Food Science, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Iran. E-mail: j_fayazi@asnrukh.ac.ir

³ Corresponding Author, Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. E-mail: m.salehi@sbmu.ac.ir

⁴ Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. E-mail: s.jajarmi@sbmu.ac.ir

https://doi.org/10.22059/jap.2024.370186.623774

Abstract

Introduction: Myostatin (MSTN) is a member of transforming growth factor-β (TGF-β), which is a negative regulator for muscle differentiation and growth in various mammals and plays a key role in muscle growth and meat quality. Today, CRISPR technology can be used to accurately change any attribute. The CRISPR/Cas9 technology creates double-strand breaks (DSBs) in the target region of DNA, which can be repaired by homology repair (HDR) in the presence of the corresponding homologous repair template or by non-homologous end joining (NHEJ).
Materials and Methods: Guide RNAs (sgRNA) were designed using CRISPOR online software. Eggs collected from 150 Varami sheep were placed in 50-microliter drops of culture medium in an incubator containing 7% CO₂ and 95% humidity. 22-24 hours after IVM, a mixture of two guide RNAs cloned in a CRISPR vector was injected into each oocyte at a concentration of 30 ng with a microinjection microscope. After microinjection, the parthenogenesis method was used to fertilize the eggs. After the activation of the eggs in the wells of the 96-well plate, the bottom of which was covered with cumulus cells and sage medium for eight days in an incubator with a temperature of 38.5 degrees Celsius and 7% CO₂gas, they were cultivated under conditions of maximum humidity. After eight days, the zygotes that had reached the embryonic stage were analyzed with a fluorescent microscope. The embryos of the test group that emitted green light, as well as one embryo from the control group, were individually placed in nine microliters of DNA Lysis to prepare their genomes. They were incubated in a temperature program of one hour at 65 degrees and ten minutes at 90 degrees. In order to investigate the gene editing of the embryos that emitted green light, the PCR products of five greened embryos along with one embryo of the control group were sequenced by the trench method.
Results and Discussion: Finally, 12 sheep embryos were produced, which were analyzed with a fluorescent microscope, and a total of five embryos emitted green light. The green light indicated that they had received the CRISPR/Cas9 technology. Among the five embryos, two of the embryos with guide RNA 1 showed a single nucleotide deletion upstream of PAM. Additionally, two of the embryos showed a single nucleotide deletion in guide RNA 2, while one of the embryos remained unchanged. Sequence analysis of the knockout embryos revealed that 83% of the cells were cut. After creating two types of single nucleotide deletions in different positions of sheep embryos, the effect of this genomic editing was detected by examining the amino acid sequence of the embryos in the control group and those carrying the mutation. It was observed that the deletion of a single nucleotide caused by guide RNA resulted in a change in the genome framework and termination code, leading to a shorter amino acid sequence in the edited sheep compared to the control group. This research marked the first time that laboratory embryos of Varamin sheep genetically manipulated by CRISPR/Cas9 technology were produced.
Conclusion: The nucleotide sequence of MSTN gene in Varami sheep was different from the sequence recorded in NCBI. Five embryos showed CRISPR technology markers. Both of the designed guide RNAs caused mutations in the nucleotide sequence and termination code in the amino acid sequence.

Keywords

References

Ahsani, M., Mohammadabadi, M., & Shamsaddini, M. (2010). Clostridium perfringens isolate typing by multiplex PCR. Journal of Venomous Animals and Toxins including Tropical Diseases, 16, 573-578.

Aiello, D., Patel, K., & Lasagna, E. (2018). The myostatin gene: an overview of mechanisms of action and its relevance to livestock animals. Animal genetics, 49(6), 505-519.

Chebo, C., Betsha, S., & Melesse, A. (2022). Chicken genetic diversity, improvement strategies and impacts on egg productivity in Ethiopia: a review. World's Poultry Science Journal, 78(3), 803-821.

Chen, M., Zhao, Y., Xu, X., Zhang, X., Zhang, J., Wu, S., Liu, Z., Yuan, Y., Guo, X., & Qi, S. (2023). AMSTN Del273C mutation withFGF5knockout sheep by CRISPR/Cas9 promotes skeletal muscle myofiber hyperplasia via MEK-ERK-FOSL1 axis.

Crispo, M., Mulet, A., Tesson, L., Barrera, N., Cuadro, F., dos Santos-Neto, P., Nguyen, T., Crénéguy, A., Brusselle, L., & Anegón, I. (2015). Efficient generation of myostatin knock-out sheep using CRISPR/Cas9 technology and microinjection into zygotes. PloS one, 10(8), e0136690.

Dilger, A. C., Gabriel, S., Kutzler, L., McKeith, F., & Killefer, J. (2010). The myostatin null mutation and clenbuterol administration elicit additive effects in mice. animal, 4 (3), 466-471.

Guo, R., Wang, H., Meng, C., Gui, H., Li, Y., Chen, F., Zhang, C., Zhang, H., Ding, Q., & Zhang, J. (2023). Efficient and Specific Generation of MSTN-Edited Hu Sheep Using C-CRISPR. Genes, 14(6), 1216.

Kalds, P., Gao, Y., Zhou, S., Cai, B., Huang, X., Wang, X., & Chen, Y. (2020). Redesigning small ruminant genomes with CRISPR toolkit: overview and perspectives. Theriogenology, 147, 25-33.

Kraemer, W. J., & Ratamess, N. A. (2005). Hormonal responses and adaptations to resistance exercise and training. Sports medicine, 35, 339-361.

Liu, G., Zhang, Y., & Zhang, T. (2020). Computational approaches for effective CRISPR guide RNA design and evaluation. Computational and structural biotechnology journal, 18, 35-44.

Lv, Q., Yuan, L., Deng, J., Chen, M., Wang, Y., Zeng, J., Li, Z., & Lai, L. (2016). Efficient generation of myostatin gene mutated rabbit by CRISPR/Cas9. Scientific reports, 6(1), 25029.

M Scharenberg, A., Duchateau, P., & Smith, J. (2013). Genome engineering with TAL-effector nucleases and alternative modular nuclease technologies. Current gene therapy, 13(4), 291-303.

Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., & Church, G. M. (2013). RNA-guided human genome engineering via Cas9. science, 339 (6), 823-826.

McPherron, A. C., & Lee, S.-J. (2002). Suppression of body fat accumulation in myostatin-deficient mice. The Journal of clinical investigation, 109(5), 595-601.

Menchaca, A., dos Santos-Neto, P., Mulet, A., & Crispo, M. (2020). CRISPR in livestock: From editing to printing. Theriogenology, 150, 247-254.

Mohammadabadi, M., Golkar, A., Askari Hesni, M., & Khezri, A. (2023). The effect of fennel (Foeniculum vulgare) on insulin-like growth factor 1 gene expression in the rumen tissue of Kermani sheep. Agricultural Biotechnology Journal, 15(4), 239-256.

Peterson, A. (2017). CRISPR: express delivery to any DNA address. Oral Diseases, 23(1), 5-11.

Roudbar, M. A., Abdollahi-Arpanahi, R., Mehrgardi, A. A., Mohammadabadi, M., Yeganeh, A. T., & Rosa, G. (2018). Estimation of the variance due to parent-of-origin effects for productive and reproductive traits in Lori-Bakhtiari sheep. Small Ruminant Research, 160, 95-102.

Safaei, S. M. H., Dadpasand, M., Mohammadabadi, M., Atashi, H., Stavetska, R., Klopenko, N., & Kalashnyk, O. (2022). An origanum majorana leaf diet influences myogenin gene expression, performance, and carcass characteristics in lambs. Animals, 13(1), 14.

Wegner, J., Albrecht, E., Fiedler, I., Teuscher, F., Papstein, H. J., & Ender, K. (2000). Growth and breed related changes of muscle fiber characteristics in cattle. Journal of animal science, 78(6), 1485-1496.

Yi, D., Zhou, S. W., Qiang, D., Bei, C., Zhao, X. E., Zhong, S., Jin, M. H., Wang, X. L., & Chen, Y. l. (2020). The CRISPR/Cas9 induces large genomic fragment deletions of MSTN and phenotypic changes in sheep. Journal of Integrative Agriculture, 19(4), 1065-1073.

Zhang, Y., Wang, Y., Yulin, B., Tang, B., Wang, M., Zhang, C., Zhang, W., Jin, J., Li, T., & Zhao, R. (2019). CRISPR/Cas9‐mediated sheep MSTN gene knockout and promote sSMSCs differentiation. Journal of cellular biochemistry, 120(2), 1794-1806.

Zhou, S., Kalds, P., Luo, Q., Sun, K., Zhao, X., Gao, Y., Cai, B., Huang, S., Kou, Q., & Petersen, B. (2022). Optimized Cas9: sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality. BMC genomics, 23(1), 348.

Animal Production

Knockout of myostatin (MSTN) gene using CRISPR/Cas9 technology in order to produce knockout Varamini sheep embryos

References

References

Volume 26, Issue 2
July 2024
Pages 99-110

Knockout of myostatin (MSTN) gene using CRISPR/Cas9 technology in order to produce knockout Varamini sheep embryos

References

References

Volume 26, Issue 2July 2024Pages 99-110

Volume 26, Issue 2
July 2024
Pages 99-110