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bioRxiv · 10.1101/2020.03.13.990630

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

Gaudelli, N.·Lam, D. K.·Rees, H. A.·Sola-Esteves, N. M.·Barrera, L. A.·Born, D. A.·Edwards, A.·Gehrke, J. M.·Lee, S.-J.·Liquori, A. J.·Murray, R.·Packer, M. S.·Rinaldi, C.·Slaymaker, I. M.·Yen, J.·Young, L. E.·Ciaramella, G.
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Abstract

The foundational adenine base editors (e.g. ABE7.10) enable programmable C*G to T*A point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG PAM sites, ABE8s result in [~]1.5x higher editing at protospacer positions A5-A7 and [~]3.2x higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a [~]4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the {gamma}-globin genes HBG1 and HBG2, with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification which is maintained when multiplexed across three loci. Delivered as mRNA, ABE8s induce no significant levels of sgRNA-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.

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Gaudelli, N., Lam, D. K., Rees, H. A., Sola-Esteves, N. M., Barrera, L. A., Born, D. A., Edwards, A., Gehrke, J. M., Lee, S.-J., Liquori, A. J., Murray, R., Packer, M. S., Rinaldi, C., Slaymaker, I. M., Yen, J., Young, L. E., Ciaramella, G.. 2020-03-16. Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application. https://doi.org/10.1101/2020.03.13.990630

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