DESIGN AND INVESTIGATION OF DUMBBELL-SHAPED DNA MINIMAL VECTORS FOR CRISPR/CAS9 EDITING
type: CreativeWork
description: We explored the use of minimalistic dumbbell-shaped DNA vectors for the delivery of the CRISPR/Cas9 system. Using three targets, i.e. glucose-6-phosphate dehydrogenase (G6PD) Mahidol, G6PD Mediterranean and Southeast Asian Ovalocytosis, and a dual reporter assay, we demonstrated that dumbbell vectors offer a viable alternative to plasmids for delivery of the CRISPR/Cas9 gene editing tool. Our analyses of sgRNA structure revealed that unstructured CRISPR RNA (crRNA) domains in sgRNA exhibited higher target cleavage activities as compared with structured crRNA domains. We also found that molecular helper functions enhancing DNA nuclear import (SV40 enhancer sequence) and Cas9 mRNA nuclear export (WPRE), improved the editing efficiency of both plasmid and dumbbell vectors. Further, we developed the first genetic 3-in-1 editing vectors comprising sgRNA and Cas9 plus a double- or single-stranded DNA repair template. We first investigated the potential impact of secondary structure formation in ssDNA RT on gene editing.
abstract: We explored the use of minimalistic dumbbell-shaped DNA vectors for the delivery of the CRISPR/Cas9 system. Using three targets, i.e. glucose-6-phosphate dehydrogenase (G6PD) Mahidol, G6PD Mediterranean and Southeast Asian Ovalocytosis, and a dual reporter assay, we demonstrated that dumbbell vectors offer a viable alternative to plasmids for delivery of the CRISPR/Cas9 gene editing tool. Our analyses of sgRNA structure revealed that unstructured CRISPR RNA (crRNA) domains in sgRNA exhibited higher target cleavage activities as compared with structured crRNA domains. We also found that molecular helper functions enhancing DNA nuclear import (SV40 enhancer sequence) and Cas9 mRNA nuclear export (WPRE), improved the editing efficiency of both plasmid and dumbbell vectors. Further, we developed the first genetic 3-in-1 editing vectors comprising sgRNA and Cas9 plus a double- or single-stranded DNA repair template. We first investigated the potential impact of secondary structure formation in ssDNA RT on gene editing.
author: AVANTIKA GHOSH
keywords: CRISPR/Cas9, Dumbbell vectors, sgRNA, editing, DNA repair, gene therapy
date published: 2019-08-16
date modified: 2026-07-24T03:32:43Z
provider: National University of Singapore
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: National University of Singapore