ESPE Abstracts (2019) 92 P1-311

Optimisation of Transfection Methods Using DNA, RNA and Protein Formats for CRISPR Cas9 Mediated Gene Knock out in Beta-TC-6 Cells.

Preetha Purushothaman1, Amy Walker1, Khalid Hussain2, Stephen Hart1

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

Background: Beta-tumour cells (βTC) are a group of highly differentiated beta cell lines derived by expression of the SV40 T antigen (Tag) oncoprotein under control of the insulin promoter in transgenic mice. βTC-6 cells exhibit glucose stimulated insulin secretion which makes them a valuable tool in understanding the mechanisms that regulate insulin secretion.

The CRISPR/Cas9 genome-editing platform is a versatile and powerful technology to efficiently create genetically engineered living cells and organisms. This system requires a complex of Cas9 endonuclease protein with a gene-targeting guide RNA (gRNA) to introduce double-strand DNA breaks (DSBs) at specific locations in the genome which are repaired by Non-Homologous End Joining (NHEJ) pathway, resulting in insertions and/or deletions (indels) which disrupt the targeted locus. The success of CRISPR genome editing experiments is limited by the intracellular delivery and expression of Cas9 protein and gRNA.

Aims: The aim of the project 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 function of the two genes of interest- ABCC8 and HADH.

Methods: Several CRISPR sgRNAs were designed to target each gene and tested to identify the best sgRNAs to generate the KO cellular models. 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.

We performed transfections 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 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, transfection conditions were optimised by varying Beta-TC-6 cell density and amount of transfection reagent. For the delivery of Cas9/gRNA as an RNP format, different non viral vectors including Lipofectamine 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 BTC6 cells.

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