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Nature. 2019 May;569(7756):433-437. doi: 10.1038/s41586-019-1161-z. Epub 2019 Apr 17.

Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.

Grünewald J1,2,3,4, Zhou R1,2,3, Garcia SP1, Iyer S1, Lareau CA1,5, Aryee MJ1,2,3,4,5, Joung JK6,7,8,9.

Author information

1
Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA.
2
Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA.
3
Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA.
4
Department of Pathology, Harvard Medical School, Boston, MA, USA.
5
Department of Biostatistics, Harvard T. H. Chan School of Public Health, Boston, MA, USA.
6
Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA. jjoung@mgh.harvard.edu.
7
Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA. jjoung@mgh.harvard.edu.
8
Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA. jjoung@mgh.harvard.edu.
9
Department of Pathology, Harvard Medical School, Boston, MA, USA. jjoung@mgh.harvard.edu.

Abstract

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.

PMID:
30995674
DOI:
10.1038/s41586-019-1161-z

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