Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair

Brett M Sansbury; Amanda M Hewes; Eric B Kmiec

doi:10.1038/s42003-019-0705-y

Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair

Commun Biol. 2019 Dec 6:2:458. doi: 10.1038/s42003-019-0705-y. eCollection 2019.

Authors

Brett M Sansbury^{1

2}, Amanda M Hewes², Eric B Kmiec^{1

2}

Affiliations

¹ 1Gene Editing Institute, Helen F. Graham Cancer Center & Research Institute, Christiana Care Health System, Newark, DE USA.
² 2Department of Medical and Molecular Sciences, University of Delaware, Newark, DE USA.

Abstract

As CRISPR-Cas systems advance toward clinical application, it is essential to identify all the outcomes of gene-editing activity in human cells. Reports highlighting the remarkable success of homology-directed repair (HDR) in the treatment of inherited diseases may inadvertently underreport the collateral activity of this remarkable technology. We are utilizing an in vitro gene-editing system in which a CRISPR-Cas complex provides the double-stranded cleavage and a mammalian cell-free extract provides the enzymatic activity to promote non-homologous end joining, micro-homology mediated end joining, and homology-directed repair. Here, we detail the broad spectrum of gene-editing reaction outcomes utilizing Cas9 and Cas12a in combination with single-stranded donor templates of the sense and nonsense polarity. This system offers the opportunity to see the range of outcomes of gene-editing reactions in an unbiased fashion, detailing the distribution of DNA repair outcomes as a function of a set of genetic tools.

Keywords: CRISPR-Cas systems; CRISPR-Cas9 genome editing; DNA damage and repair.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Base Sequence
CRISPR-Cas Systems*
DNA Repair*
Gene Editing*
Gene Targeting
Genetic Variation*
Humans