CRISPR–Cas base-editor technology enables targeted nucleotide alterations, and is being increasingly used for research and potential therapeutic applications1,2. The most widely used cytosine base editors (CBEs) induce deamination of DNA cytosines using the rat APOBEC1 enzyme, which is targeted by a linked Cas protein–guide RNA complex3,4. Previous studies of the specificity of CBEs have identified off-target DNA edits in mammalian cells5,6. Here we show that a CBE with rat APOBEC1 can cause extensive transcriptome-wide deamination of RNA cytosines in human cells, inducing tens of thousands of C-to-U edits with frequencies ranging from 0.07% to 100% in 38–58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, and 5′ and 3′ untranslated region mutations. We engineered two CBE variants bearing mutations in rat APOBEC1 that substantially decreased the number of RNA edits (by more than 390-fold and more than 3,800-fold) in human cells. These variants also showed more precise on-target DNA editing than the wild-type CBE and, for most guide RNAs tested, no substantial reduction in editing efficiency. Finally, we show that an adenine base editor7 can also induce transcriptome-wide RNA edits. These results have implications for the use of base editors in both research and clinical settings, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.
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Plasmids encoding the most relevant constructs shown in this work, including both SECURE BE3 variants, have been deposited to Addgene (http://www.addgene.org/browse/article/28197497/; Addgene IDs 123611–123616).
All RNA-seq data used in this study have been deposited in the Gene Expression Omnibus (GEO) repository (National Center for Biotechnology Information). The files are accessible through the GEO Series accession number GSE121668. All WES and targeted amplicon sequencing data have been deposited at the SRA repository under bioproject number PRJNA497753. All other relevant data are available from the corresponding author on request.
The authors will make all previously unreported custom computer code used in this work available upon reasonable request.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
J.K.J., J.G. and R.Z. are supported by the Defense Advanced Research Projects Agency (HR0011-17-2-0042). Support was also provided by the National Institutes of Health (RM1 HG009490 to J.K.J. and J.G. and R35 GM118158 to J.K.J. and M.J.A.). J.K.J. is additionally supported by the Desmond and Ann Heathwood MGH Research Scholar Award. We thank M. M. Kaminski, B. P. Kleinstiver and K. Petri for discussions; V. Pattanayak for input on the manuscript; Y. E. Tak, G. Boulay, M. K. Clement, A. A. Sousa, R. T. Walton, M. L. Bobbin, M. V. Maus and A. Schmidts for technical advice; and P. K. Cabeceiras and O. R. Cervantes for technical assistance. J.K.J. dedicates this paper to the memory of C. J. Park.