Complex

Validated clonal edits

We analyse selected clones with in-house bioinformatics software, before sending the final clonal cell line for full NGS analysis to confirm the edit before shipping.

CRISPR edited iPSCs

Our automated CRISPR engineering platform can reliably deliver CRISPR edited iPSC lines for advanced disease modelling.

OXGENE has considerable in-house expertise in genetically engineering mammalian cells as well as access to the latest CRISPR editing technology.

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Complex modifications

Many genetic diseases are the result of more than one gene knockout or other types of mutations entirely, and culturing primary cells from patients with these mutations can be very difficult. With the support of our automated gene editing platform, our in-house experts generate clonal cell lines modelling complex disease scenarios, including double knock-outs, conditional knock-outs, knock-ins, gene fusions and chromosomal rearrangements.

We’ve also optimised our automated gene editing platform to accommodate high throughput edits of inducible Pluripotent Stem Cells (iPSCs). These are a particularly powerful tool for disease modelling and drug discovery, but can be challenging to work with, as they differentiate in response to cellular stress, and exhibit notoriously low editing efficiency. However, we’ve optimised our gene editing workflow to achieve a careful balance between cellular stability and editing efficiency to deliver reliably edited iPSC lines.

We’ve successfully engineered a number of chromosomal translocations using CRISPR/Cas9. Here, we’ve shown RT-PCR (upper panels) and sequencing data (lower panels) confirming gene fusions between (from left to right); Ewing sarcoma breakpoint region 1 (EWSR1) and the ETS transcription factor (FLI1), EMAP like 4 (EML4) and ALK receptor tyrosine kinase (ALK), coiled-coil domain containing 6 (CCDC6) and ROS1, and ETS variant 6 (ETV6) and ABL proto-oncogene 1, non receptor tyrosine kinase (ABL1).

Illustration of the genetic engineering strategy we employed to delete approximately a 1Mb region between genes A and B, resulting in a gene fusion of approximately 1.5Kb between these two genes. RT-PCR data from pooled untransfected cells (-/-) and transfected samples before (+/-) and after (+/+) puromycin pulse enrichment. The band isn’t present after puromycin selection due to the absence of the puromycin resistence gene. Sequencing the remaining 1.5Kb amplicon confirmed deletion of the 1Mb region; the promoter from Gene X is now fused to the 3’UTR of Gene Y.

We put a strong emphasis on communication with our partners, who remain in direct contact with both the project manager and scientific team throughout the project.

Find out more about bespoke complex CRISPR engineering projects for disease modelling.

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