한빛사 논문
Do Yon Kim1,2,†, Su Bin Moon1,2,†, Jeong-Heon Ko1,2, Yong-Sam Kim1,2,3,* and Daesik Kim1,*
1Genome Editing Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea, 2KRIBB School of Bioscience, Korea University of Science and Technology (UST) , Daejeon 34141, Republic of Korea and 3GenKOre, Daejeon 34141, Republic of Korea
*To whom correspondence should be addressed.
†The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.
Abstract
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.
논문정보
TOP52020년 후보
관련 링크
연구자 키워드
연구자 ID
관련분야 연구자보기
소속기관 논문보기
관련분야 논문보기