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

• Published in: Nature biotechnology Vol. 37; no. 4; pp. 430 - 435
• Main Authors: Kim, Daesik, Kim, Da-eun, Lee, Gyeorae, Cho, Sung-Ik, Kim, Jin-Soo
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.04.2019; Nature Publishing Group
• Subjects: 631/1647; 631/1647/1513; 631/45/147; 631/61/201/2110; Adenine; Adenine - metabolism; Agriculture; Aminohydrolases - metabolism; Animals; Base Sequence; Binding Sites - genetics; Bioinformatics; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Cells (Biology); Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas systems; Cytosine; Cytosine Deaminase - metabolism; Deamination; Deoxyribonucleic acid; DNA; DNA Breaks, Double-Stranded; DNA glycosylase; DNA sequencing; Editors; Endonuclease; Endonuclease V; Endonuclease VIII; Gene editing; Gene Editing - methods; Gene mutation; Gene sequencing; Genetic aspects; Genomes; Genomics; Guanine; HEK293 Cells; Humans; In vitro methods and tests; Kinases; Letter; Life Sciences; Methods; Nucleases; Plant cells; Proteins; Purines; Pyrimidines; Ribonucleic acid; Ribonucleoproteins; RNA; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Target recognition; Whole Genome Sequencing
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/s41587-019-0050-1

Adenine base editors 1 enable efficient targeted adenine-to-guanine single nucleotide conversions to induce or correct point mutations in human cells, animals, and plants 1 – 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 sequencing 5 – 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 specificities 6 . Using targeted sequencing, we also show that use of preassembled adenine base editor ribonucleoproteins, modified guide RNAs 5 , 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.