ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2019) 66 P5 | DOI: 10.1530/endoabs.66.P5

Optimisation of transfection methods using various formats of gRNA delivery for CRISPR Cas9 mediated gene knock out in Beta-TC-6 cells

Preetha Purushothaman1, Amy Walker1, Martin Attwood1, Ruhina Maeshima1, Khalid Hussain2 & Stephen Hart1

1UCL GREAT ORMOND STREET INSTITUTE OF CHILD HEALTH, London, UK; 2Sidra Medical and Research Center, Doha, Qatar

Background: The CRISPR/Cas9 genome-editing platform is a powerful technology to create genetically engineered cells and organisms. However, the success of CRISPR genome editing experiments is limited by the intracellular delivery and expression of Cas9 endonuclease protein and guide RNA (gRNA). Beta-tumour cells (βTC-6), derived from transgenic mice, exhibit glucose stimulated insulin secretion which makes them a valuable tool in understanding the mechanisms that regulate insulin secretion.

Aims: The aim was to identify the optimal transfection conditions for the intracellular delivery of Cas9 protein and gRNA in βTC-6 cells so as to create a KO mouse cell model of Congenital Hyperinsulinism (CHI). Such cellular models would play a key role in the elucidation of the molecular mechanisms underlying CHI.

Methods: gRNAs were designed to target two genes of interest- Abcc8 and Hadh. Optimisation of the delivery of CRISPR/Cas9 system included the evaluation of different formats such as plasmid DNA, mRNA and RNP complex using a reporter gene. Transfections were performed using different combinations of molecules including: plasmid DNA, Cas9 protein and gRNA in an RNP format to maximize targeting of the Abcc8 and Hadh gene in βTC-6 cells. A reporter (GFP)was initially used to evaluate the transfection efficiency of the plasmid DNA and mRNA with flow cytometry and fluorescent microscopy being used to detect the GFP signal. To obtain the highest transfection efficiency, conditions were optimised by varying cell density and amount of transfection reagent. For the delivery of Cas9/gRNA as an RNP format, different non-viral vectors including Lipofectamine 2000 and nanocomplexes were used. At the molecular level, the disruption of the gene was confirmed by Sanger sequencing and T7 ENDO assay.

Results: Progress so far has addressed the optimisation of transfection conditions to deliver CRISPR/Cas9 in βTC-6 cells. Determination of editing efficiency using the ICE tool by Synthego revealed a low KO score.

Future work: Transfection by electroporation using synthetic sgRNA pre-complexed to the Cas protein in the ribonucleoprotein (RNP) format may improve editing efficiencies.

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