Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing.
Nucleic Acids Res
; 49(2): 969-985, 2021 01 25.
Article
in En
| MEDLINE
| ID: mdl-33398341
Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Ribonucleoproteins
/
Parvovirinae
/
DNA End-Joining Repair
/
Recombinational DNA Repair
/
CRISPR-Cas Systems
/
Gene Editing
/
Genetic Vectors
Limits:
Adult
/
Humans
Language:
En
Journal:
Nucleic Acids Res
Year:
2021
Document type:
Article
Affiliation country:
China
Country of publication:
United kingdom