Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2017) 51 P024 | DOI: 10.1530/endoabs.51.P024

BSPED2017 Poster Presentations Miscellaneous/other (16 abstracts)

Using CRISPR/Cas9 gene editing to study molecular mechanisms of congenital hyperinsulinism

Preetha Purushothaman 1 , Ahmad Aldossary 1 , Stephen Hart 1 & Khalid Hussain 2

1Institute of Child Health, University College London, London, UK; 2Sidra Medical and Research Center, Doha, Qatar.

Background: Congenital hyperinsulinism (CHI) is a heterogeneous genetically determined condition that is characterised by unregulated secretion of insulin from pancreatic β-cells. The most common and severe cases are caused by mutations in the ABCC8 gene encoding the SUR1 subunit of the KATP channel subunit. Autosomal recessive mutations in HADH gene are a rare cause of CHI. The advances in CRISPR/Cas9 gene editing technology has enabled the induction of targeted modifications in a variety of model organisms. However, the applicability and efficiency of CRISPR/Cas9 gene editing as a tool to unravel the molecular mechanisms that lead to the unregulated insulin secretion in CHI has been little reported.

Methods: Single-guide RNAs (sgRNA) were designed using three different web tools for predicting on-target and off-target probabilities. The exons 1, 3 and 6 were targeted in the ABCC8 gene while exons 1,3 and 4 were selected in the HADH gene in MIN6 cell lines. sgRNAs were then cloned into the plasmid vector pX330 encoding the S. pyogenes Cas9 endonuclease (SpCas9) gene and transfected into the MIN6 cells. The resulting double-strand breaks were repaired by the non-homologous end joining (NHEJ) technique. Gene editing efficiency was determined by the T7 Endonuclease I mutation detection assay and Sanger sequencing.

Results: Six sgRNAs have been designed of which two have targeted the ABCC8 gene at two sites within exons 3 and 6. We demonstrated insertion and deletions (indels) within the genomic DNA of the ABCC8 gene which shows the potential generation of a knock-out mutation in the ABCC8 gene of MIN6 cells using the CRISPR/Cas9 gene editing technique.

Conclusions: The results of our study so far has demonstrated the potential of the use of Cas9/gRNA system as an efficient reverse genetic tool in studying the molecular mechanisms underlying CHI. Our future aims are to: conduct further molecular interrogation to confirm the KO in ABCC8 gene; create a KO allele of HADH gene in the MIN6 cell line and further, use the newly generated KO mutant cells, to analyse the function of these genes.

[Grant MR/M023265/1]

Volume 51

45th Meeting of the British Society for Paediatric Endocrinology and Diabetes

British Society for Paediatric Endocrinology and Diabetes 

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