Cytosine but not adenine base editor generates mutations in mice

Abstract

Deaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms and its ultimate success therapeutically depends on its accuracy. Here we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editor (ABE) in mouse embryos using unbiased whole genome sequencing of a family-based trio cohort. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compared to ABE-edited mice and controls.

Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity

“…WGS analysis has been the gold standard for direct, thorough, and unbiased assessment of unexpected off-target edits by Cas proteins and base editors (Iyer et al, 2018;Tang et al, 2018;Willi et al, 2018;Jin et al, 2019;Li et al, 2019;Zuo et al, 2019;Lee et al, 2020). However, its major drawbacks, namely lowthroughput, expense, and length have prevented this approach from being used widely (Iyer et al, 2018;Li et al, 2019;Willi et al, 2018).…”

“…WGS methods for evaluating genome-wide off-target events via the CRISPR-Cas system or base editors are low throughput, costly, and time-consuming (Iyer et al, 2018;Tang et al, 2018;Willi et al, 2018;Jin et al, 2019;Li et al, 2019;Zuo et al, 2019;Lee et al, 2020). In contrast, the orthogonal R-loop assay reported by Doman et al (2020) has proven effective in evaluating sgRNA-independent off-target activity in human cells.…”

“…Current CBEs, such as BE3, which fuse the nickase-type Cas9 (nCas9) protein with a deaminase domain and an uracil glycosylase inhibitor (UGI), catalyze conversions of cytosine to thymine at the guide RNA (gRNA)-targeted sites (Gaudelli et al, 2017;Komor et al, 2016;Nishida et al, 2016). Previous studies using whole-genome sequencing (WGS) showed that BE3 induces off-target C-to-T changes in rice, mouse, and human cells (Jin et al, 2019;Lee et al, 2020;McGrath et al, 2019;Zuo et al, 2019). These mutations were independent of single-guide RNA (sgRNA)-Cas9-programmed DNA binding, and were enriched in transcribed regions of the genome (Jin et al, 2019;Zuo et al, 2019).…”

“…These mutations were independent of single-guide RNA (sgRNA)-Cas9-programmed DNA binding, and were enriched in transcribed regions of the genome (Jin et al, 2019;Zuo et al, 2019). They are probably due to the high affinity of the cytosine deaminases for singlestranded DNA (ssDNA) (Jin et al, 2019;Lee et al, 2020;Zuo et al, 2019). This high affinity also affects the precision of ontarget activity, such that most CBEs generate multiple C mutations if multiple cytosines are present within or around target sites (Cheng et al, 2019;Gehrke et al, 2018;Kim et al, 2017;Lee et al, 2018;Tan et al, 2020).…”

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

“…As with all base editors, ABE8s have the potential to act at off-target loci in the genome and transcriptome 1,2,16,17,[36][37][38][39][40][41] . Guided by prior publications on this topic, we undertook an extensive assessment of the off-target deamination effects of ABE8s.…”