Optimisation of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 : single-guide RNA (sgRNA) delivery system in a goat model
Yu Huang A B , Yige Ding A , Yao Liu A , Shiwei Zhou A , Qiang Ding A , Hailong Yan A , Baohua Ma C , Xiaoe Zhao C , Xiaolong Wang
A and Yulin Chen A D
+ Author Affiliations
- Author Affiliations
A Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
B Biomanufacturing Engineering Laboratory, Advanced Manufacturing Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China.
C College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling 712100, China.
D Corresponding author. Email: chenyulin@nwafu.edu.cn
Reproduction, Fertility and Development 31(9) 1533-1537 https://doi.org/10.1071/RD18485
Submitted: 19 September 2018 Accepted: 20 March 2019 Published: 13 May 2019
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an efficient method for the production of gene-edited animals. We have successfully generated gene-modified goats and sheep via zygote injection of Cas9 mRNA and single-guide RNA (sgRNA) mixtures. However, the delivery system for microinjection largely refers to methods established for mice; optimised injection conditions are urgently required for the generation of large animals. Here, we designed a study to optimise the Cas9 mRNA and sgRNA delivery system for goats. By comparing four computational tools for sgRNA design and validating the targeting efficiency in goat fibroblasts, we suggest a protocol for the selection of desirable sgRNAs with higher targeting efficiency and negligible off-target mutations. We further evaluated the editing efficiency in goat zygotes injected with Cas9 : sgRNA (sg8) and found that injection with 50 ng μL−1 Cas9 mRNA and 25 ng μL−1 sgRNA yielded an increased editing efficiency. Our results provide a reference protocol for the optimisation of the injection conditions for the efficient editing of large animal genomes via the zygote injection approach.
Additional keywords: microinjection, genome editing, delivery system, gene targeting.
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