한빛사 논문
Daesik Kim1,4, Kayeong Lim1,2,4, Sang-Tae Kim2, Sun-heui Yoon1, Kyoungmi Kim2, Seuk-Min Ryu1,2 & Jin-Soo Kim1-3
1Department of Chemistry, Seoul National University, Seoul, Republic of Korea. 2Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, Republic of Korea. 3Department of Functional Genomics, University of Science and Technology, Daejeon, Republic of Korea. 4These authors contributed equally to this work.
Correspondence to : Jin-Soo Kim or Daesik Kim
Abstract
Cas9-linked deaminases, also called base editors, enable targeted mutation of single nucleotides in eukaryotic genomes. However, their off-target activity is largely unknown. Here we modify digested-genome sequencing (Digenome-seq) to assess the specificity of a programmable deaminase composed of a Cas9 nickase (nCas9) and the deaminase APOBEC1 in the human genome. Genomic DNA is treated with the base editor and a mixture of DNA-modifying enzymes in vitro to produce DNA double-strand breaks (DSBs) at uracil-containing sites. Off-target sites are then computationally identified from whole genome sequencing data. Testing seven different single guide RNAs (sgRNAs), we find that the rAPOBEC1?nCas9 base editor is highly specific, inducing cytosine-to-uracil conversions at only 18 ± 9 sites in the human genome for each sgRNA. Digenome-seq is sensitive enough to capture off-target sites with a substitution frequency of 0.1%. Notably, off-target sites of the base editors are often different from those of Cas9 alone, calling for independent assessment of their genome-wide specificities.
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