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Genome-wide target specificity of CRISPR RNA-guided adenine base editors

Cited 62 time in Web of Science Cited 69 time in Scopus
Authors
Kim, Daesik; Kim, Da-eun; Lee, Gyeorae; Cho, Sung-Ik; Kim, Jin-Soo
Issue Date
2019-04
Citation
Nature Biotechnology, Vol.37 No.4, pp.430-435
Abstract
Adenine base editors(1) enable efficient targeted adenine-to-guanine single nucleotide conversions to induce or correct point mutations in human cells, animals, and plants(1-4). Here we present a modified version of Digenome-seq, an in vitro method for identifying CRISPR (clustered regularly interspaced short palindromic repeats)-induced double-strand breaks using whole-genome sequencings(5-8), to assess genomewide target specificity of adenine base editors. To produce double-strand breaks at sites containing inosines, the products of adenine deamination, we treat human genomic DNA with an adenine base editor 7.10 protein-guide RNA complex and either endonuclease V or a combination of human alkyladenine DNA glycosylase and endonuclease VIII in vitro. Digenome-seq detects adenine base editor off-target sites with a substitution frequency of 0.1% or more. We show that adenine base editor 7.10, the cytosine base editor BE3, and unmodified CRISPR-associated protein 9 (Cas9) often recognize different off-target sites, highlighting the need for independent assessments of their genome-wide specificities(6). Using targeted sequencing, we also show that use of preassembled adenine base editor ribonucleoproteins, modified guide RNAs5,8-11, and Sniper/Cas9 (ref.(12)) reduces adenine base editor off-target activity in human cells.
ISSN
1087-0156
URI
https://hdl.handle.net/10371/165712
DOI
https://doi.org/10.1038/s41587-019-0050-1
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