Unbiased investigation of specificities of prime editing systems in human cells

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Unbiased investigation of specificities of prime editing systems in human cells
Do Yon Kim; Su Bin Moon; Jeong Heon KoYong Sam Kim; Daesik Kim
Bibliographic Citation
Nucleic Acids Research, vol. 48, no. 18, pp. 10576-10589
Publication Year
Prime editors (PEs) enable targeted precise editing, including the generation of substitutions, insertions and deletions, in eukaryotic genomes. However, their genome-wide specificity has not been explored. Here, we developed Nickase-based Digenome-seq (nDigenome-seq), an in vitro assay that uses whole-genome sequencing to identify single-strand breaks induced by CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nickase. We used nDigenome-seq to screen for potential genome-wide off-target sites of Cas9 H840A nickase, a PE component, targeted to nine human genomic sites. Then, using targeted amplicon sequencing of off-target candidates identified by nDigenome-seq, we showed that only five off-target sites showed detectable PE-induced modifications in cells, at frequencies ranging from 0.1 to 1.9%, suggesting that PEs provide a highly specific method of precise genome editing. We also found that PE specificity in human cells could be further improved by incorporating mutations from engineered Cas9 variants, particularly eSpCas9 and Sniper Cas9, into PE.
Oxford Univ Press
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Synthetic Biology and Bioengineering Research Institute > Genome Editing Research Center > 1. Journal Articles
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