Enhancing prime editor flexibility with coiled-coil heterodimers.
Genome Biol
; 25(1): 108, 2024 04 26.
Article
in En
| MEDLINE
| ID: mdl-38671524
ABSTRACT
BACKGROUND:
Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency.RESULTS:
Herein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship. We demonstrate that the CC-PE maintains or even surpasses the efficiency of unsplit PE in installing intended edits, with no increase in the levels of undesired byproducts within tested loci amongst a variety of cell types (HEK293T, A549, HCT116, and U2OS). Furthermore, coiled-coil heterodimers are used to engineer SpCas9-NG-PE and SpRY-PE, two Cas9 variants with more flexible editing scope. Similarly, the resulting NG-CC-PE and SpRY-CC-PE also achieve equivalent or enhanced efficiency of precise editing compared to the intact PE. When the dual AAV vectors carrying CC-PE are delivered into mice to target the Pcsk9 gene in the liver, CC-PE enables highly efficient precise editing, resulting in a significant reduction of plasma low-density lipoprotein cholesterol and total cholesterol.CONCLUSIONS:
Our innovative, modular system enhances flexibility, thus potentially facilitating the in vivo applicability of prime editing.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Gene Editing
Limits:
Animals
/
Humans
Language:
En
Journal:
Genome Biol
Journal subject:
BIOLOGIA MOLECULAR
/
GENETICA
Year:
2024
Document type:
Article
Affiliation country:
China