Efficient base editing for multiple genes and loci in pigs using base editors

Jingke Xie,Xiaoyu Yang,Zhen Ouyang,Yu Zhao,Nan Li,Weikai Ge,Kepin Wang,Quanjun Zhang,Yanhui Liang,Qishuai Liu,Han Wu,Longquan Quan,Xiaoping Huang ,Ting Lan,Fangbing Chen,Xiaoping Li,Jin Qiu ,Zhaoming Liu,Hui Shi,Chengdan Lai,Nana Fan,Shixue Gou,Liangxue Lai

doi:10.1038/s41467-019-10421-8

Abstract

Cytosine base editors (CBEs) enable programmable C-to-T conversion without DNA double-stranded breaks and homology-directed repair in a variety of organisms, which exhibit great potential for agricultural and biomedical applications. However, all reported cases only involved C-to-T substitution at a single targeted genomic site. Whether C-to-T substitution is effective in multiple sites/loci has not been verified in large animals. Here, by using pigs, an important animal for agriculture and biomedicine, as the subjective animal, we showed that CBEs could efficiently induce C-to-T conversions at multiple sites/loci with the combination of three genes, including DMD, TYR, and LMNA, or RAG1, RAG2, and IL2RG, simultaneously, at the embryonic and cellular levels. CBEs also could disrupt genes (pol gene of porcine endogenous retrovirus) with dozens of copies by introducing multiple premature stop codons. With the CBEs, pigs carrying single gene or multiple gene point mutations were generated through embryo injection or nuclear transfer approach.

Highlights

Cytosine base editors (CBEs) enable programmable C-to-T conversion without DNA doublestranded breaks and homology-directed repair in a variety of organisms, which exhibit great potential for agricultural and biomedical applications
To confirm whether base editing for multiple sites can be achieved by BE3 in the genome of porcine embryos (Fig. 1a), we selected six porcine genes, namely, Duchenne muscular dystrophy (DMD), TYR, LMNA, RAG1, RAG2, and IL2RG, which encode dystrophin, tyrosinase, lamin A/C, RAG1 protein, RAG2 protein, and IL-2 receptor gamma chain, respectively, as the target sites (Fig. 1b)
Premature stop codons (Q493STOP and Q28STOP) would be generated by a single C-toT conversion at the target sites in DMD and TYR, which are expected to result in Duchenne muscular dystrophy (DMD) and albinism, respectively

Summary

Introduction

Cytosine base editors (CBEs) enable programmable C-to-T conversion without DNA doublestranded breaks and homology-directed repair in a variety of organisms, which exhibit great potential for agricultural and biomedical applications. With the CBEs, pigs carrying single gene or multiple gene point mutations were generated through embryo injection or nuclear transfer approach. The newly developed base editors (BEs)[8,9,10] efficiently enable precise and highly predictable nucleotide substitutions (C-to-T or A-to-G conversion) at targeted genomic loci, independent of DSBs and donor templates. These BEs have great potential applications for both agriculture and biomedicine. With the CBE system, we achieve gene editing in pigs with single gene point mutation as well as multiple gene point mutations through either embryo injection or somatic cell nuclear transfer (SCNT) approach

Methods

Results

Conclusion