Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Pivot Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Target Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Query: EC:3.5.1.4 (
deaminase
)
5,113
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE-
PACE
) to improve their editing efficiency and target sequence compatibility. We used BE-
PACE
to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1
deaminase
, while maintaining efficient editing in all other sequence contexts tested. Another evolved
deaminase
, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1
deaminase
with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between
deaminase
activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.
...
PMID:Continuous evolution of base editors with expanded target compatibility and improved activity. 3140 30
Applications of adenine base editors (ABEs) have been constrained by the limited compatibility of the deoxyadenosine deaminase component with Cas homologs other than SpCas9. We evolved the
deaminase
component of ABE7.10 using phage-assisted non-continuous and continuous evolution (PANCE and
PACE
), which resulted in ABE8e. ABE8e contains eight additional mutations that increase activity (k
app
) 590-fold compared with that of ABE7.10. ABE8e offers substantially improved editing efficiencies when paired with a variety of Cas9 or Cas12 homologs. ABE8e is more processive than ABE7.10, which could benefit screening, disruption of regulatory regions and multiplex base editing applications. A modest increase in Cas9-dependent and -independent DNA off-target editing, and in transcriptome-wide RNA off-target editing can be ameliorated by the introduction of an additional mutation in the TadA-8e domain. Finally, we show that ABE8e can efficiently install natural mutations that upregulate fetal hemoglobin expression in the BCL11A enhancer or in the the HBG promoter in human cells, targets that were poorly edited with ABE7.10. ABE8e augments the effectiveness and applicability of adenine base editing.
...
PMID:Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. 3243 48
