PMID- 31976379
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20240328
IS  - 2398-502X (Print)
IS  - 2398-502X (Electronic)
IS  - 2398-502X (Linking)
VI  - 4
DP  - 2019
TI  - A CRISPR/Cas9 genome editing pipeline in the EndoC-betaH1 cell line to study genes 
      implicated in beta cell function.
PG  - 150
LID - 10.12688/wellcomeopenres.15447.2 [doi]
LID - 150
AB  - 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-betaH1). 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 (ATP) channel gene expression in contrast to siRNA silencing. 
      Overall, this strategy to efficiently create stable KO in the human beta cell 
      line EndoC-betaH1 will allow for a better understanding of genes involved in beta 
      cell dysfunction, their underlying functional mechanisms and T2D pathogenesis.
CI  - Copyright: (c) 2020 Grotz AK et al.
FAU - Grotz, Antje K
AU  - Grotz AK
AUID- ORCID: 0000-0002-7736-510X
AD  - Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, 
      Oxford, OX3 7LE, UK.
FAU - Abaitua, Fernando
AU  - Abaitua F
AD  - Wellcome Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK.
FAU - Navarro-Guerrero, Elena
AU  - Navarro-Guerrero E
AUID- ORCID: 0000-0001-8076-1480
AD  - Target Discovery Institute, University of Oxford, Oxford, OX3 7FZ, UK.
FAU - Hastoy, Benoit
AU  - Hastoy B
AUID- ORCID: 0000-0003-1244-7857
AD  - Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, 
      Oxford, OX3 7LE, UK.
FAU - Ebner, Daniel
AU  - Ebner D
AD  - Target Discovery Institute, University of Oxford, Oxford, OX3 7FZ, UK.
FAU - Gloyn, Anna L
AU  - Gloyn AL
AUID- ORCID: 0000-0003-1205-1844
AD  - Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, 
      Oxford, OX3 7LE, UK.
AD  - Wellcome Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK.
AD  - Oxford NIHR Biomedical Research Centre, Churchill Hospital, Oxford, OX3 7LE, UK.
LA  - eng
GR  - MR/L020149/1/MRC_/Medical Research Council/United Kingdom
GR  - U01 DK085545/DK/NIDDK NIH HHS/United States
GR  - U01 DK105535/DK/NIDDK NIH HHS/United States
GR  - WT_/Wellcome Trust/United Kingdom
GR  - 200837/Z/16/Z/WT_/Wellcome Trust/United Kingdom
PT  - Journal Article
DEP - 20200429
PL  - England
TA  - Wellcome Open Res
JT  - Wellcome open research
JID - 101696457
PMC - PMC6961417
OTO - NOTNLM
OT  - CRISPR/Cas9
OT  - EndoC-betaH1
OT  - NEUROD1
OT  - Type 2 Diabetes
OT  - gene knockout
OT  - human beta cells
OT  - insulin secretion
COIS- Competing interests: ALG has received honoraria from Novo Nordisk and Merck.
EDAT- 2020/05/13 06:00
MHDA- 2020/05/13 06:01
PMCR- 2020/04/29
CRDT- 2020/05/13 06:00
PHST- 2020/04/22 00:00 [accepted]
PHST- 2020/05/13 06:00 [entrez]
PHST- 2020/05/13 06:00 [pubmed]
PHST- 2020/05/13 06:01 [medline]
PHST- 2020/04/29 00:00 [pmc-release]
AID - 10.12688/wellcomeopenres.15447.2 [doi]
PST - epublish
SO  - Wellcome Open Res. 2020 Apr 29;4:150. doi: 10.12688/wellcomeopenres.15447.2. 
      eCollection 2019.