DNA repair

Enzyme

Base pair

Endonuclease

Nucleotide

Biology

Guide RNA

Gene

Genome editing

Cas9

Genome

Deamination

Human genome

Cytosine

DNA glycosylase

Guanine

Computational biology

CRISPR

Genetics

Biochemistry

Nature Biotechnology

Adenine base editors1 enable efficient targeted adenine-to-guanine single nucleotide conversions to induce or correct point mutations in human cells, animals, and plants1–4. 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 sequencing5–8, to assess genome-wide 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 specificities6. 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. Unbiased detection of off-target editing by adenine base editors in vitro uncovers differences in the specificity patterns of adenine and cytosine base editors, and of unmodified Cas9.

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