Li, C, Fu, Z, Yang, Y et al. (8 more authors) (2018) CRISPR/Cas9-mediated editing of GABRR2 gene in RGC-5 cells induces random exon deletion, exon splicing and new exon recruitment. Biochemical Engineering Journal, 139. pp. 43-50. ISSN 1369-703X
Abstract
CRISPR/Cas9 and its variations provide an efficient tool for targeted genome editing. CRISPR/Cas9-based genome editing can induce mutations in genome that has high probability to cause exon truncation or deletion. However, screening mutations in diploid cells is difficult because of two copies of chromosome. It is an important task for determining genotypes in diploid cells subjected to editing before using these cells in gene function study. In this study, we applied CRISPR/Cas9 to edit the GABRR2 gene in mouse retinal ganglion cells to study what exactly happened in two alleles and what real mRNA isoforms formed in diploid cells. A single sgRNA was employed to generate double-strand DNA breaks. PCR sequencing was used for single clone validation in diploid cells subjected to editing. The indels and the corresponding effects at the target locus were further studied at genomic and RNA levels. We observed that CRISPR/Cas9 induces random deletions in the target region of GABRR2 gene, and both big and small indels can lead to unexpected high probability of exon truncation/skipping. In addition, random deletions in genomic region recruited introns to generate new “exon”. It is the first observation of exon recruitment by CRISPR/Cas9-mediated GABRR2 gene editing. The observations may offer a reference for the future gene splicing study.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2018 Elsevier B.V. This is an author produced version of a paper published in Biochemical Engineering Journal. Uploaded in accordance with the publisher's self-archiving policy. |
Keywords: | CRISPR-Cas9; GABRR2; RGC-5; Exon skipping; Exon recruitment |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 08 Oct 2018 16:15 |
Last Modified: | 09 Aug 2019 00:43 |
Status: | Published |
Publisher: | Elsevier |
Identification Number: | 10.1016/j.bej.2018.08.005 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:136500 |