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Cytosine and Adenine Base Editors Expressed from Messenger RNAs Mediate Efficient Base Corrections In Vitro and In Vivo

Authors: Anton McCaffrey1, Jordana M Henderson1, Gregory A Newby2,3,4, Tingting Jiang5, Mike Houston1, Julie Powers1, Wen Xue5, and David Liu2,3,4

1 TriLink Biotechnologies LLC, San Diego, CA, USA
2 Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA
3 Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA
4 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA
5 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA

New tools for genome editing raise the possibility of precisely correcting genetic defects. Zinc-finger nucleases, TALENs, or CRISPR-Cas9 create double stranded breaks that stimulate homologous recombination with a donor DNA. However, these approaches are inefficient and give rise to undesired indels. Here, we use transient mRNA treatment to introduce permanent single base edits mediated by base editors. Cytosine base editors (CBEs) use a Cas9 nickase fused to a cytosine deaminase and uracil DNA glycosylase inhibitor. C:G base pairs are converted to T:A pairs with high efficiency and minimal indels. Similarly, adenine base editors (ABEs) use an evolved deoxyadenosine deaminase fused to Cas9 nickase to convert A:T base pairs to C:G pairs. Editing efficiencies of >90% were observed without cell sorting. In contrast to viral vectors and plasmids, mRNA offers key advantages including 1) reduced risk of vector integration; 2) ability to edit hard-to-transfect, non-dividing cells; 3) ability to repeat administer in vivo; and 4) transient expression to maximize specificity. Here, we compare sequence-optimized, chemically-modified CBE and ABE mRNAs in HEK293 cells. Western blot analysis showed higher expression of 5-methoxyuridine modified, sequence optimized mRNAs compared to unmodified mRNA. HPLC purified wild type and N1-methylpseudouridine modified editors were also tested. In cultured cells, mRNA resulted in higher editing frequencies than plasmid vectors. We also demonstrate the ability to simultaneously edit multiple sites with one base editor mRNA, and edit previously inaccessible genomic sites. Finally, we developed a mouse model by editing mouse zygotes with injected mRNA encoding BE4max variant mRNAs. This model will be used to test in vivo ABE corrections in future studies.

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