Precise A\u2022T to G\u2022C base editing in the zebrafish genome

Wei Qin,Yunxing Liu,Song Li,Xiaochan Lu,Haipeng Bai,Shuo Lin

doi:10.1186/s12915-018-0609-1

Wei Qin, Yunxing Liu + Show 4 more

Open Access

https://doi.org/10.1186/s12915-018-0609-1

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Abstract

BackgroundBase editors are a class of genome editing tools with the ability to efficiently induce point mutations in genomic DNA, without inducing double-strand breaks or relying on homology-direct repair as in other such technologies. Recently, adenine base editors (ABEs) have been developed to mediate the conversion of A•T to G•C in genomic DNA of human cells, mice, and plants. Here, we investigated the activity and efficiency of several adenine base editors in zebrafish and showed that base editing can be used to create new models of pathogenic diseases caused by point mutations.ResultsThe original ABE7.10 exhibits almost no activity in zebrafish. After codon optimization, we found that a zABE7.10 variant could induce targeted conversion of adenine to guanine in zebrafish at multiple tested genomic loci, and all the target sites showed a high rate of germline targeting efficiency. Furthermore, using this system, we established a zebrafish model of 5q-Syndrome that contained a new point mutation in rps14. The further modification of zABE7.10 by a bipartite nuclear localization signals (bpNLS) resulted in 1.96-fold average improvement in ABE-mediated editing efficiency at four sites.ConclusionsCollectively, this system, designated as zABE7.10, provides a strategy to perform A•T to G•C base editing in zebrafish and enhances its capacity to model human diseases.

Highlights

Base editors are a class of genome editing tools with the ability to efficiently induce point mutations in genomic DNA, without inducing double-strand breaks or relying on homology-direct repair as in other such technologies
Codon-optimized zABE7.10 could induce base editing in zebrafish To explore whether the pCMV-ABE7.10-gRNA complex can mediate A to G conversion in zebrafish genome (Fig. 1a), we selected gRNAs for five target sites that have been previously shown to be efficient in zebrafish using BE or Target-AID system (Fig. 1b) [5, 15]
Sequencing analysis indicated that the pCMV-ABE7.10 system did not induce base conversion at any of the selected loci (Fig. 1c)

Summary

Introduction

Base editors are a class of genome editing tools with the ability to efficiently induce point mutations in genomic DNA, without inducing double-strand breaks or relying on homology-direct repair as in other such technologies. In order to complement the HDR method, one approach called “base editing” was developed, which can irreversibly convert C to T at target loci without double-strand break (DSB) [2, 3] Among these base editors, the third-generation base editor (BE3) has been applied in several species, including zebrafish [4,5,6]. To further improve these base editors, strategies have been developed to expand editing window [7], increase editing specificity and efficiency [8, 9], improve base excision repair inhibition [10], and change protospacer-adjacent motif (PAM) compatibilities [7] These base editors only mediate the conversion of C to T, which reduces its application in gene editing

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