How to find the right target?

Finding the right target is crucial for any type of drug discovery research. Therefore, modifications of genomic targets via insertion of mutations or knockouts for target identification is a widely used method to identify protein function and possible targets for drug development.

Target validation in IPF (idiopathic pulmonary fibrosis)

Preferably, results are generated in a biologically relevant background e.g. with primary cells, displaying the closest to nature conditions for an in vitro experiment. With the rise of CRISPR technology and the availability of high-throughput electroporation technologies, modifications of all genes of a certain pathway, or a number of genes linked to a specific disease, can be achieved even in primary cell and diseased cell backgrounds. Matteo Martufi et al. from the GSK Target Science and Fibrosis Discovery Performance Unit were able to generate single and double knockouts with a genome editing efficiency of up to 100% in a single transfection procedure in primary human diseased lung fibroblasts, confirming the involvement of Smad 3 but not Smad 2 in disease progression in IPF. The group employed the CRISPR-Cas9 technology in use with RNPs to generate the gene specific knockouts in one single transfection step.

In this study, the researcher tested several pulse parameters with the Nucleofector^® System and found out, that the combination of pulse code CM-138 and solution P3, such as the addition of an electroporation enhancer, showed the best conditions for genome editing. For further information, please check the detailed protocol in the supplementary part of the published article. In a second step, bulk cell populations with > 90% indels were used one week after Nucleofection for the Scar-in-a-Jar phenotypic assay, which measures type I collagen deposition after TGF-β stimulation.

If you are looking for a method for target identification, then the 4D-Nucleofector^® 96-well Unit  or the 384-well Nucleofector^® System is the perfect choice. You can use these for CRISPR screens even in hard-to-transfect and diseased primary cells like IPF Human Lung Fibroblasts as the systems show high transfection efficiencies and are quite easy and fast for testing several sgRNAs. 

So, feel free to check our Knowledge Center for already existing optimized nucleofection protocols of specific cell lines and primary cells. Further, you can get in touch with our scientific support team to plan your upcoming transfection experiments. 

The only question is: What’s your next target ?

Written by

Camilla
Scientific Support Specialist