Directed evolution of adenine base editors with increased activity and therapeutic application.
Nat Biotechnol
; 38(7): 892-900, 2020 07.
Article
in En
| MEDLINE
| ID: mdl-32284586
ABSTRACT
The foundational adenine base editors (for example, ABE7.10) enable programmable Aâ¢T to Gâ¢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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Adenine
/
RNA, Guide, Kinetoplastida
/
Cytosine
/
CRISPR-Cas Systems
Limits:
Humans
Language:
En
Journal:
Nat Biotechnol
Journal subject:
BIOTECNOLOGIA
Year:
2020
Document type:
Article
Affiliation country: