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      CRISPR DNA base editors with reduced RNA off-target and self-editing activities

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          Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations 14 . However, we recently demonstrated that they can also induce widespread guide RNA-independent RNA base edits 5 and created SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that have reduced unwanted RNA editing activity 5 . Here, we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA editing and comparable on-target DNA activities that are also among the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A (hA3A)-based CBE induces substantial RNA base edits, whereas an enhanced A3A (eA3A)-CBE 6 , human activation-induced cytidine deaminase (hAID)-CBE 7 , and the petromyzon marinus cytidine deaminase (pmCDA1)-based CBE Target-AID 4 induce reduced RNA edits. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base editor coding sequences.

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          Most cited references 6

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          Simplified mammalian DNA isolation procedure.

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            Crystal structure of Staphylococcus aureus tRNA adenosine deaminase TadA in complex with RNA.

            Bacterial tRNA adenosine deaminases (TadAs) catalyze the hydrolytic deamination of adenosine to inosine at the wobble position of tRNA(Arg2), a process that enables this single tRNA to recognize three different arginine codons in mRNA. In addition, inosine is also introduced at the wobble position of multiple eukaryotic tRNAs. The genes encoding these deaminases are essential in bacteria and yeast, demonstrating the importance of their biological activity. Here we report the crystallization and structure determination to 2.0 A of Staphylococcus aureus TadA bound to the anticodon stem-loop of tRNA(Arg2) bearing nebularine, a non-hydrolyzable adenosine analog, at the wobble position. The cocrystal structure reveals the basis for both sequence and structure specificity in the interactions of TadA with RNA, and it additionally provides insight into the active site architecture that promotes efficient hydrolytic deamination.
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              tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli.

              We report the characterization of tadA, the first prokaryotic RNA editing enzyme to be identified. Escherichia coli tadA displays sequence similarity to the yeast tRNA deaminase subunit Tad2p. Recombinant tadA protein forms homodimers and is sufficient for site-specific inosine formation at the wobble position (position 34) of tRNA(Arg2), the only tRNA having this modification in prokaryotes. With the exception of yeast tRNA(Arg), no other eukaryotic tRNA substrates were found to be modified by tadA. How ever, an artificial yeast tRNA(Asp), which carries the anticodon loop of yeast tRNA(Arg), is bound and modified by tadA. Moreover, a tRNA(Arg2) minisubstrate containing the anticodon stem and loop is sufficient for specific deamination by tadA. We show that nucleotides at positions 33-36 are sufficient for inosine formation in mutant Arg2 minisubstrates. The anticodon is thus a major determinant for tadA substrate specificity. Finally, we show that tadA is an essential gene in E.coli, underscoring the critical function of inosine at the wobble position in prokaryotes.

                Author and article information

                Nat Biotechnol
                Nat. Biotechnol.
                Nature biotechnology
                25 July 2019
                02 September 2019
                September 2019
                02 March 2020
                : 37
                : 9
                : 1041-1048
                [1 ]Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA, USA
                [2 ]Center for Cancer Research and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA
                [3 ]Department of Pathology, Harvard Medical School, Boston, MA, USA
                [4 ]Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA
                [5 ]These authors contributed equally to this work
                Author notes

                Author Contributions

                All wet lab experiments were performed by R.Z. and J.G. S.I., C.A.L., S.P.G. and M.J.A. performed computational analysis of the data. J.G. and J.K.J. conceived of and designed the study. J.G., M.J.A. and J.K.J. supervised the work. J.G. and J.K.J. wrote the initial manuscript draft and all authors contributed to the writing of the final manuscript.

                [* ]Correspondence should be addressed to: jjoung@

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