Programmable Base Editing of the Sheep Genome Revealed No Genome-Wide Off-Target Mutations.

Shiwei Zhou,Yao Liu,Chong He,Wenzhi Niu,Qiang Ding,Jiao Liu,Qifang Kou,Ying Wang,Yige Ding,Chao Li,Bei Cai,Xiaolong Wang,Tad Sonstegard,Bjoern Petersen,Xiaoe Zhao,Baohua Ma,Yulin Chen,Honghao Yu,Guanwei Li

doi:10.3389/fgene.2019.00215

Abstract

Since its emergence, CRISPR/Cas9-mediated base editors (BEs) with cytosine deaminase activity have been used to precisely and efficiently introduce single-base mutations in genomes, including those of human cells, mice, and crop species. Most production traits in livestock are induced by point mutations, and genome editing using BEs without homology-directed repair of double-strand breaks can directly alter single nucleotides. The p.96R > C variant of Suppressor cytokine signaling 2 (SOCS2) has profound effects on body weight, body size, and milk production in sheep. In the present study, we successfully obtained lambs with defined point mutations resulting in a p.96R > C substitution in SOCS2 by the co-injection of BE3 mRNA and a single guide RNA (sgRNA) into sheep zygotes. The observed efficiency of the single nucleotide exchange in newborn animals was as high as 25%. Observations of body size and body weight in the edited group showed that gene modification contributes to enhanced growth traits in sheep. Moreover, targeted deep sequencing and unbiased family trio-based whole genome sequencing revealed undetectable off-target mutations in the edited animals. This study demonstrates the potential for the application of BE-mediated point mutations in large animals for the improvement of production traits in livestock species.

Highlights

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 is widely used to establish site-specific genome-edited cell lines and animal models (Sander and Joung, 2014)
The called Single-nucleotide polymorphisms (SNPs) were filtered according to the following criteria: (1) SNPs that were identified by both Genome Analysis Toolkit (GATK) and SAMtools; (2) excluding SNPs that exist in NCBI sheep SNP database (>59 million SNPs); (3) excluding SNPs that exist in our sheep SNP database (n = 294, >79 million SNPs1); (4) within the remaining SNPs, SNPs with C and G converted to other base types were selected
Five mated Tan sheep that were treated for superovulation received 54 one-cell fertilized oocytes; after 53 embryos were subjected to micro-injection, 20 developing embryos were transplanted into eight recipients

Summary

INTRODUCTION

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 is widely used to establish site-specific genome-edited cell lines and animal models (Sander and Joung, 2014). After several generations of modification, base editor 3 (BE3) including rAPOBEC1, nCas (A840H), Single Base Editing in Domestic Sheep and uracil DNA glycosylase inhibitor (UGI) was developed; the mutation efficiency was up to 74.9% in mammalian cells (Komor et al, 2016). We recently reported the usage of the BE3 system to induce nonsense mutations in the goat FGF5 gene, to generate animals with longer hair fibers (Li G. et al, 2018). It was the first base editing study in large animals and further inspired us to examine the feasibility of induce amino acid exchanges in sheep. We used a parent-progeny whole genome sequencing (WGS) approach to show that no off-target mutations were detected and the mutation frequency in edited animals is equivalent to that in control groups

MATERIALS AND METHODS

Design of sgRNA

RESULTS AND DISCUSSION

CONCLUSION