A CRISPR/Cas9 genome editing pipeline in the EndoC-βH1 cell line to study genes implicated in beta cell function.
| Autor: | Grotz AK; Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, Oxford, OX3 7LE, UK., Abaitua F; Wellcome Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK., Navarro-Guerrero E; Target Discovery Institute, University of Oxford, Oxford, OX3 7FZ, UK., Hastoy B; Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, Oxford, OX3 7LE, UK., Ebner D; Target Discovery Institute, University of Oxford, Oxford, OX3 7FZ, UK., Gloyn AL; Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, Oxford, OX3 7LE, UK.; Wellcome Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK.; Oxford NIHR Biomedical Research Centre, Churchill Hospital, Oxford, OX3 7LE, UK. |
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| Jazyk: | angličtina |
| Zdroj: | Wellcome open research [Wellcome Open Res] 2020 Apr 29; Vol. 4, pp. 150. Date of Electronic Publication: 2020 Apr 29 (Print Publication: 2019). |
| DOI: | 10.12688/wellcomeopenres.15447.2 |
| Abstrakt: | Type 2 diabetes (T2D) is a global pandemic with a strong genetic component, but most causal genes influencing the disease risk remain unknown. It is clear, however, that the pancreatic beta cell is central to T2D pathogenesis. In vitro gene-knockout (KO) models to study T2D risk genes have so far focused on rodent beta cells. However, there are important structural and functional differences between rodent and human beta cell lines. With that in mind, we have developed a robust pipeline to create a stable CRISPR/Cas9 KO in an authentic human beta cell line (EndoC-βH1). The KO pipeline consists of a dual lentiviral sgRNA strategy and we targeted three genes ( INS , IDE , PAM ) as a proof of concept. We achieved a significant reduction in mRNA levels and complete protein depletion of all target genes. Using this dual sgRNA strategy, up to 94 kb DNA were cut out of the target genes and the editing efficiency of each sgRNA exceeded >87.5%. Sequencing of off-targets showed no unspecific editing. Most importantly, the pipeline did not affect the glucose-responsive insulin secretion of the cells. Interestingly, comparison of KO cell lines for NEUROD1 and SLC30A8 with siRNA-mediated knockdown (KD) approaches demonstrate phenotypic differences. NEUROD1- KO cells were not viable and displayed elevated markers for ER stress and apoptosis. NEUROD1 -KD, however, only had a modest elevation, by 34%, in the pro-apoptotic transcription factor CHOP and a gene expression profile indicative of chronic ER stress without evidence of elevated cell death. On the other hand, SLC30A8 -KO cells demonstrated no reduction in K Competing Interests: Competing interests: ALG has received honoraria from Novo Nordisk and Merck. (Copyright: © 2020 Grotz AK et al.) |
| Databáze: | MEDLINE |
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