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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Vydáno v:Nature biotechnology Ročník 38; číslo 7; s. 892 - 900
Hlavní autoři: 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
Médium: Journal Article
Jazyk:angličtina
Vydáno: United States Nature Publishing Group 01.07.2020
Témata:
Adenine
Adenine - metabolism
Adenosine
Adenosine Deaminase
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
Loci
Lymphocytes
Lymphocytes T
mRNA
Mutation
Mutation - genetics
Ribonucleic acid
RNA
RNA, Guide, CRISPR-Cas Systems
ISSN:1087-0156, 1546-1696, 1546-1696
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Shrnutí: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.
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ISSN:1087-0156
1546-1696
1546-1696
DOI:10.1038/s41587-020-0491-6