Targeting fidelity of adenine and cytosine base editors in mouse embryos

Hye Kyung Lee,Shannon M Miller,Chengyu Liu,Lothar Hennighausen,Michaela Willi,David R Liu,Sojung Kim

doi:10.1038/s41467-018-07322-7

Abstract

Base editing directly converts a target base pair into a different base pair in the genome of living cells without introducing double-stranded DNA breaks. While cytosine base editors (CBE) and adenine base editors (ABE) are used to install and correct point mutations in a wide range of organisms, the extent and distribution of off-target edits in mammalian embryos have not been studied in detail. We analyze on-target and proximal off-target editing at 13 loci by a variety of CBEs and ABE in more than 430 alleles generated from mouse zygotic injections using newly generated and published sequencing data. ABE predominantly generates anticipated A•T-to-G•C edits. Among CBEs, SaBE3 and BE4, result in the highest frequencies of anticipated C•G-to-T•A products relative to editing byproducts. Together, these findings highlight the remarkable fidelity of ABE in mouse embryos and identify preferred CBE variants when fidelity in vivo is critical.

Highlights

Base editing directly converts a target base pair into a different base pair in the genome of living cells without introducing double-stranded DNA breaks
Our analysis reveals the remarkable fidelity of adenine base editors (ABE) use in mouse embryos and identifies preferred cytosine base editors (CBE) variants when fidelity in vivo is paramount, findings important to the use of base editing in mouse genetics in basic and translational research
We investigated mutations in 436 mutant alleles - 222 alleles edited by CBE variants, and 214 alleles edited by ABE and performed statistical analysis (Supplementary Fig. 1)

Summary

Introduction

Base editing directly converts a target base pair into a different base pair in the genome of living cells without introducing double-stranded DNA breaks. Its application in reliably and efficiently introducing defined mutations into the mouse genome might be limited as double-stranded DNA breaks created by Cas[9] routinely result in non-homologous end joining (NHEJ) repair and the insertion and deletion (indel) of sequences at target sites[7,8]. Base editing[9,10], a newer form of genome editing, directly converts target CG base pairs to TA, or target AT base pairs to GC, without inducing double-stranded DNA breaks[11], which should make it less likely to cause undesired mutations, such as deletions or insertions. Our analysis reveals the remarkable fidelity of ABE use in mouse embryos and identifies preferred CBE variants when fidelity in vivo is paramount, findings important to the use of base editing in mouse genetics in basic and translational research

Methods

Results

Conclusion