Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration.
J Biomed Sci
; 31(1): 32, 2024 Mar 26.
Article
in En
| MEDLINE
| ID: mdl-38532479
ABSTRACT
BACKGROUND:
The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR-Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency.METHOD:
Here we report the development of a multicolour fluorescence assay for studying CRISPR-Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR-Cas9 strategies.RESULT:
We found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration.CONCLUSION:
Our results highlight the need for a more stringent assessment of CRISPR-Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.Key words
Full text:
1
Database:
MEDLINE
Main subject:
CRISPR-Cas Systems
/
Gene Editing
Limits:
Humans
Language:
En
Journal:
J Biomed Sci
Journal subject:
MEDICINA
Year:
2024
Type:
Article
Affiliation country:
United kingdom