Electroporation Delivery of Cas9 sgRNA Ribonucleoprotein-Mediated Genome Editing in Sheep IVF Zygotes
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Published:2024-08-23
Issue:17
Volume:25
Page:9145
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ISSN:1422-0067
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Container-title:International Journal of Molecular Sciences
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language:en
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Short-container-title:IJMS
Author:
Pi Wenhui1, Feng Guangyu1, Liu Minghui1, Nie Cunxi1ORCID, Chen Cheng1ORCID, Wang Jingjing2, Wang Limin2, Wan Pengcheng2, Liu Changbin2, Liu Yi3, Zhou Ping2
Affiliation:
1. College of Animal Science and Technology, Shihezi University, Shihezi 832003, China 2. State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China 3. State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China
Abstract
The utilization of electroporation for delivering CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In this study, our research focused on targeting the ACTG1 and MSTN genes in sheep, revealing a threshold phenomenon in electroporation with a voltage tolerance in sheep in vitro fertilization (IVF) zygotes. Various poring voltages near 40 V and pulse durations were examined for electroporating sheep zygotes. The study concluded that stronger electric fields required shorter pulse durations to achieve the optimal conditions for high gene mutation rates and reasonable blastocyst development. This investigation also assessed the quality of Cas9/sgRNA ribonucleoprotein complexes (Cas9 RNPs) and their influence on genome editing efficiency in sheep early embryos. It was highlighted that pre-complexation of Cas9 proteins with single-guide RNA (sgRNA) before electroporation was essential for achieving a high mutation rate. The use of suitable electroporation parameters for sheep IVF zygotes led to significantly high mutation rates and heterozygote ratios. By delivering Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) to zygotes through electroporation, targeting the MSTN (Myostatin) gene, a knock-in efficiency of 26% was achieved. The successful generation of MSTN-modified lambs was demonstrated by delivering Cas9 RNPs into IVF zygotes via electroporation.
Funder
Startup Foundation for Advanced Talents of Shihezi university Bingtuan Science and Technology Project Science and Technology Specialist Team Service Program of Shihezi University Research Training Program for Undergraduates of Shihezi University
Reference40 articles.
1. Recent advances in the genomic resources for sheep;Woolley;Mamm. Genome,2023 2. Kalds, P., Zhou, S., Cai, B., Liu, J., Wang, Y., Petersen, B., Sonstegard, T., Wang, X., and Chen, Y. (2019). Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era. Front. Genet., 10. 3. CRISPR technology: A decade of genome editing is only the beginning;Wang;Science,2023 4. Wang, X., Niu, Y., Zhou, J., Yu, H., Kou, Q., Lei, A., Zhao, X., Yan, H., Cai, B., and Shen, Q. (2016). Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep. Sci. Rep., 6. 5. Crispo, M., Mulet, A.P., Tesson, L., Barrera, N., Cuadro, F., dos Santos-Neto, P.C., Nguyen, T.H., Crénéguy, A., Brusselle, L., and Anegón, I. (2015). Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes. PLoS ONE, 10.
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