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 journalArticle

89 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
Externally publishedYes

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
RNA
Genes
Genome
Uracil
Human Genome
DNA
Guide RNA
Double-Stranded DNA Breaks
Deoxyribonuclease I
Cytosine
Nucleotides
Mutation
Substitution reactions
Enzymes

ASJC Scopus subject areas

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

Cite this

Kim, D., Lim, K., Kim, S. T., Yoon, S. H., Kim, K., Ryu, S. M., & Kim, J. S. (2017). Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nature Biotechnology, 35(5), 475-480. https://doi.org/10.1038/nbt.3852

Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. / Kim, Daesik; Lim, Kayeong; Kim, Sang Tae; Yoon, Sun Heui; Kim, Kyoungmi; Ryu, Seuk Min; Kim, Jin Soo.

In: Nature Biotechnology, Vol. 35, No. 5, 01.05.2017, p. 475-480.

Research output: Contribution to journalArticle

Kim, D, Lim, K, Kim, ST, Yoon, SH, Kim, K, Ryu, SM & Kim, JS 2017, 'Genome-wide target specificities of CRISPR RNA-guided programmable deaminases', Nature Biotechnology, vol. 35, no. 5, pp. 475-480. https://doi.org/10.1038/nbt.3852
Kim, Daesik ; Lim, Kayeong ; Kim, Sang Tae ; Yoon, Sun Heui ; Kim, Kyoungmi ; Ryu, Seuk Min ; Kim, Jin Soo. / Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. In: Nature Biotechnology. 2017 ; Vol. 35, No. 5. pp. 475-480.
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