Directed evolution of adenine base editors with increased activity and therapeutic application

The foundational adenine base editors (for example, ABE7.10) enable programmable A•T to G•C 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 protospac...

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Bibliographic Details
Published in:	Nature biotechnology Vol. 38; no. 7; pp. 892 - 900
Main Authors:	Gaudelli, Nicole M., Lam, Dieter K., Rees, Holly A., Solá-Esteves, Noris M., Barrera, Luis A., Born, David A., Edwards, Aaron, Gehrke, Jason M., Lee, Seung-Joo, Liquori, Alexander J., Murray, Ryan, Packer, Michael S., Rinaldi, Conrad, Slaymaker, Ian M., Yen, Jonathan, Young, Lauren E., Ciaramella, Giuseppe
Format:	Journal Article
Language:	English
Published:	New York Nature Publishing Group US 01-07-2020 Nature Publishing Group
Subjects:	631/61/338/469 631/61/51/2314 Adenine Adenine - metabolism Adenosine Adenosine Deaminase Agriculture Bioinformatics Biomedical and Life Sciences Biomedical Engineering/Biotechnology Biomedicine Biotechnology CD34 antigen CRISPR-Cas Systems - genetics Cytosine - metabolism Deamination Deoxyribonucleic acid Directed evolution DNA DNA - genetics Editing Evolution Fetuses Gene Editing - methods HEK293 Cells Hemoglobin Humans Levels Life Sciences Loci Lymphocytes Lymphocytes T mRNA Mutation Mutation - genetics Physiological aspects Ribonucleic acid RNA RNA, Guide, CRISPR-Cas Systems United States
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Summary:	The foundational adenine base editors (for example, ABE7.10) enable programmable A•T to G•C 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 protospacer adjacent motif (PAM) sites, ABE8s result in ~1.5× higher editing at protospacer positions A5–A7 and ~3.2× 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 γ-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 messenger RNA, ABE8s induce no significant levels of single guide RNA (sgRNA)-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA. Adenine base editors are evolved to be more efficient and more compatible with Cas9 variants.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1087-0156 1546-1696
DOI:	10.1038/s41587-020-0491-6