Journal
NATURE BIOTECHNOLOGY
Volume 35, Issue 5, Pages 475-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.3852
Keywords
-
Categories
Funding
- Institute for Basic Science [IBS-R021-D1]
- ToolGen, Inc. [0409-20160107]
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available