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Sci Rep. 2016 Sep 9;6:32681. doi: 10.1038/srep32681.

Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides.

Author information

1
Gene Editing Institute, Helen F. Graham Cancer Center and Research Institute, Newark, Delaware, United States of America.
2
Department of Medical Laboratory Science, College of Health Sciences, University of Delaware, Newark, Delaware, United States of America.
3
Nemours Center for Childhood Cancer Research, Alfred I. duPont Hospital for Children, Wilmington, Delaware, United States of America.
4
Department of Chemistry, Delaware State University, Dover, Delaware, United States of America.

Abstract

The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9.

PMID:
27609304
PMCID:
PMC5016854
DOI:
10.1038/srep32681
[Indexed for MEDLINE]
Free PMC Article

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