Abstract
Simple SummaryCRISPR/Cas9 driven multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to DNA double-strand breaks at multiple loci simultaneously. To overcome this problem in porcine cells we utilized Target-AID, a base editing system, to edit multiple loci in the porcine genome. We showed that the Target-AID system works well in porcine fibroblasts with up to 63.15% efficiency. This is the first report demonstrating that the Target-AID system works well in porcine cells and can be used to generate genome-edited pigs.Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3–5 base editing windows 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.
Highlights
The development of programmable nucleases, including the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, has significantly increased the efficiency of genetic engineering of livestock and has ushered in a new era in animal biotechnology
Because porcine endogenous retrovirus (PERV) is integrated into the porcine genome, it is impossible to remove this virus from pigs even when they are maintained in a microorganism-barrier facility
To avoid the risk of genotoxicity induced by multiplex double-strand breaks (DSBs) we used a Target-AID-based baseediting system to inactivate PERV in the porcine genome
Summary
The development of programmable nucleases, including the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, has significantly increased the efficiency of genetic engineering of livestock and has ushered in a new era in animal biotechnology. Conventional genome-editing systems, such as CRISPR/Cas, rely on DNA repair mechanisms after target-specific double-strand breaks (DSBs) in DNA. These systems are very efficient, they are limited in their use as a multiplex genome engineering tools because simultaneous DSBs at multiple loci induce genotoxicity and chromosomal rearrangements [6]. This disadvantage of conventional genome-editing systems for multiplex genome engineering can be overcome using the base-editing system. We targeted multiple loci in the gag and pol genes of porcine endogenous retrovirus (PERV) in the porcine genome using the Target-AID system and successfully induced target-specific base substitution
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