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Open AccessArticle

The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template

1
Gene Editing Institute, Helen F. Graham Cancer Center & Research Institute, Christiana Care Health System, Newark, DE 19713, USA
2
Department of Medical and Molecular Sciences, University of Delaware, Newark, DE 19716, USA
*
Author to whom correspondence should be addressed.
Genes 2020, 11(10), 1160; https://doi.org/10.3390/genes11101160
Received: 25 June 2020 / Revised: 22 September 2020 / Accepted: 28 September 2020 / Published: 30 September 2020
(This article belongs to the Special Issue Genes at Ten)
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing systems have enabled molecular geneticists to manipulate prokaryotic and eukaryotic genomes with greater efficiency and precision. CRISPR/Cas provides adaptive immunity in bacterial cells by degrading invading viral genomes. By democratizing this activity into human cells, it is possible to knock out specific genes to disable their function and repair errors. The latter of these activities requires the participation of a single-stranded donor DNA template that provides the genetic information to execute correction in a process referred to as homology directed repair (HDR). Here, we utilized an established cell-free extract system to determine the influence that the donor DNA template length has on the diversity of products from CRISPR-directed gene editing. This model system enables us to view all outcomes of this reaction and reveals that donor template length can influence the efficiency of the reaction and the categories of error-prone products that accompany it. A careful measurement of the products revealed a category of error-prone events that contained the corrected template along with insertions and deletions (indels). Our data provides foundational information for those whose aim is to translate CRISPR/Cas from bench to bedside. View Full-Text
Keywords: symmetrical homology arms; single-stranded donor template; homology directed repair; CRISPR/Cas12a; CRISPR/Cas9; gene editing symmetrical homology arms; single-stranded donor template; homology directed repair; CRISPR/Cas12a; CRISPR/Cas9; gene editing
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MDPI and ACS Style

Hewes, A.M.; Sansbury, B.M.; Kmiec, E.B. The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template. Genes 2020, 11, 1160. https://doi.org/10.3390/genes11101160

AMA Style

Hewes AM, Sansbury BM, Kmiec EB. The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template. Genes. 2020; 11(10):1160. https://doi.org/10.3390/genes11101160

Chicago/Turabian Style

Hewes, Amanda M.; Sansbury, Brett M.; Kmiec, Eric B. 2020. "The Diversity of Genetic Outcomes from CRISPR/Cas Gene Editing is Regulated by the Length of the Symmetrical Donor DNA Template" Genes 11, no. 10: 1160. https://doi.org/10.3390/genes11101160

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