Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos

Abstract

Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single nucleotide polymorphism in individuals. Here, we developed a method named GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole genome sequences of progeny cells of edited vs. non-edited blastomeres at E14.5 showed that off-target single nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. In contrast, cytosine base editing induced SNVs with over 20-fold higher frequencies, requiring a solution to address its fidelity.

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

“…Moreover, GOTI can evaluate off‐targets in the cell population derived from a single gene‐edited blastomere. GOTI could be useful for examining the off‐target effects of various gene‐editing tools without the interference of SNPs present in different individuals …”

“…In base editing system, off‐target can result from gRNA dependent or gRNA independent editing events . A variety of strategies have been widely used for decreasing gRNA‐dependent off‐target base editing, for instance the incorporation of mutations that enhance the specificity of DNA into the Cas9 component of base editors (BEs), adding 5′‐guanosine nucleotides to the sgRNA, or delivering the BEs as a ribonucleoprotein (RNP) complex .…”

“…A variety of strategies have been widely used for decreasing gRNA‐dependent off‐target base editing, for instance the incorporation of mutations that enhance the specificity of DNA into the Cas9 component of base editors (BEs), adding 5′‐guanosine nucleotides to the sgRNA, or delivering the BEs as a ribonucleoprotein (RNP) complex . Off‐target editing based on gRNA‐independent arises by Cas9‐independent mannered binding to the deaminase domain of a BE to C or A bases …”

In situ CRISPR‐Cas9 base editing for the development of genetically engineered mouse models of breast cancer

“…Two recent papers have shown that the CBE off-target mutation rate is higher than previously anticipated (Jin et al, 2019;Zuo et al, 2019). Although off-target activity of CBEs can be detrimental for therapeutic applications, it is much less of an issue for tumor acceleration studies in mouse models, in which random mutations collaborate with the genetically engineered mutations in driving tumorigenesis.…”

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

“…Hence, the currently developed CBEs may have trouble in sgRNA design for genes which are very short or with an extremely low GC content. Furthermore, two recent independent studies simultaneously demonstrated that the off‐target rate of BE3‐based cytosine base editor was substantially higher than that of Cas9 and ABE7.10*‐based adenosine base editor in mice and rice . The off‐target issue of CBE has also been found in bacteria.…”