Format

Send to

Choose Destination
J Vis Exp. 2017 Aug 25;(126). doi: 10.3791/56195.

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells.

Author information

1
Gene Editing Institute, Helen F. Graham Cancer Center and Research Institute, Christiana Care Health Services; Department of Medical Sciences, University of Delaware.
2
Gene Editing Institute, Helen F. Graham Cancer Center and Research Institute, Christiana Care Health Services; Department of Medical Sciences, University of Delaware; eric.b.kmiec@christianacare.org.

Abstract

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

PMID:
28872131
PMCID:
PMC5614406
DOI:
10.3791/56195
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for MyJove Corporation Icon for PubMed Central
Loading ...
Support Center