Preprint Article Version 1 This version is not peer-reviewed

Electroporation Delivery of Cas9 sgRNA ribonucleoprotein-Mediated Genome Editing in Sheep IVF Zygotes

Wenhui Pi
* ,
Guangyu Feng
,
Minghui Liu
,
Cunxi Nie
,
Cheng Chen
,
Jingjing Wang
,
Limin Wang
,
Pengcheng Wan
,
Changbin Liu
,
Ping Zhou
* ,
Yi Liu
*
Version 1 : Received: 18 July 2024 / Approved: 19 July 2024 / Online: 19 July 2024 (12:07:12 CEST)

How to cite: Pi, W.; Feng, G.; Liu, M.; Nie, C.; Chen, C.; Wang, J.; Wang, L.; Wan, P.; Liu, C.; Zhou, P.; Liu, Y. Electroporation Delivery of Cas9 sgRNA ribonucleoprotein-Mediated Genome Editing in Sheep IVF Zygotes. Preprints 2024, 2024071610. https://doi.org/10.20944/preprints202407.1610.v1 Pi, W.; Feng, G.; Liu, M.; Nie, C.; Chen, C.; Wang, J.; Wang, L.; Wan, P.; Liu, C.; Zhou, P.; Liu, Y. Electroporation Delivery of Cas9 sgRNA ribonucleoprotein-Mediated Genome Editing in Sheep IVF Zygotes. Preprints 2024, 2024071610. https://doi.org/10.20944/preprints202407.1610.v1

Abstract

The utilization of electroporation for delivering the CRISPR/Cas9 system components has enabled efficient gene editing in mammalian zygotes, facilitating the development of genome-edited animals. In our study，This 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 40V 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. The 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.

Keywords

electroporation; gene editing; CRISPR/Cas9; sheep zygotes; mutation efficiency; homologous recombination; in vitro fertilization (IVF)

Subject

Biology and Life Sciences, Animal Science, Veterinary Science and Zoology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Download PDF

Comments (0)

We encourage comments and feedback from a broad range of readers. See criteria for comments and our Diversity statement.

Leave a public comment
Send a private comment to the author(s)
* All users must log in before leaving a comment
Views 0
Downloads 0
Comments 0
Metrics 0
Get PDF Cite Share 0
Bookmark BibSonomy Mendeley Reddit Delicious
×
Alerts
Notify me about updates to this article or when a peer-reviewed version is published.
We use cookies on our website to ensure you get the best experience.
Read more about our cookies here.