Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos
By Erwei Zuo, Yidi Sun, Wu Wei, Tanglong Yuan, Wenqin Ying, Hao Sun, Liyun Yuan, Lars M. Steinmetz, Yixue Li, Hui Yang. Science, 2019
691 citations recorded by the source corpus.
Spotting off-targets from gene editing Unintended genomic modifications limit the potential therapeutic use of gene-editing tools. Available methods to find off-targets generally do not work in vivo or detect single-nucleotide changes. Three papers in this issue report new methods for monitoring gene-editing tools in vivo (see the Perspective by Kempton and Qi). Wienert et al. followed the recruitment of a DNA repair protein to DNA breaks induced by CRISPR-Cas9, enabling unbiased detection of off-target editing in cellular and animal models. Zuo et al. identified off-targets without the interference of natural genetic heterogeneity by injecting base editors into one blastomere of a two-cell mouse embryo and leaving the other genetically identical blastomere unedited. Jin et al. performed whole-genome sequencing on individual, genome-edited rice plants to identify unintended mutations. Cytosine, but not adenine, base editors induced numerous single-nucleotide variants in both mouse and rice. Science, this issue p. 286, p. 289, p. 292; see also p. 234 Cytosine base editing in mouse embryos generates numerous off-target single-nucleotide variants. 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 and nonedited blastomeres at embryonic day 14.5 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. By contrast, cytosine base editing induced SNVs at more than 20-fold higher frequencies, requiring a solution to address its fidelity.
Subfields: Genetics, Molecular Biology
View Paper: https://www.semanticscholar.org/paper/a5cfd1606bad15ea860e73195913ce5580c49e12
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In Molecular Biology
Citations in the corpus
- Cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity (2020, methodology): “NGS data were analyzed by performing four general steps: (1) Illumina demultiplexing, (2) read trimming and filtering, (3) alignment of all reads to the expected amplicon sequence, and (4) generation of alignment statistics and quantification of editing rates.”