Genome-wide target specificities of CRISPR RNA-guided programmable deaminases

Daesik Kim, Kayeong Lim, Sang Tae Kim, Sun Heui Yoon, Kyoungmi Kim, Seuk Min Ryu, Jin Soo Kim

Research output: Contribution to journalArticlepeer-review

114 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)475-480
Number of pages6
JournalNature Biotechnology
Volume35
Issue number5
DOIs
Publication statusPublished - 2017 May 1

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Biomedical Engineering

Fingerprint Dive into the research topics of 'Genome-wide target specificities of CRISPR RNA-guided programmable deaminases'. Together they form a unique fingerprint.

Cite this