Heterologously expressed targets in stable cell lines are powerful tools for discovering small molecule and antibody drug leads. Assays from these cell lines are robust, reliable and target-specific. Before moving drug candidates into animal models and human trials, translational in vitro assays are much desired intermediate steps. These assays utilize endogenously expressed targets in physiologically relevant cells. However, the challenge is target-specificity. Since related proteins from the same family as the target of interest may signal in the same pathways, the results can be confounding from these native cells. This is where gene-editing is needed.
The possibility of generating isogenically CRISPR-modified native stable cell lines along with the wildtype or parental cells are opening up a myriad of possibilities for assay development. Assays from these paired cell lines are properly controlled to pinpoint the target-specific effect. They enhance the biology relevance in cell-based assays without losing data quality. Consequently, they can serve to bridge in vitro and in vivo studies, impacting target discovery, target validation, and drug discovery screening by saving time and cost.
Multispan’s highly differentiated MULTISCREENTM CRISPR gene-editing platform uniquely combines expansive expertise in assay development, cell engineering and cloning. Like all other MULTISCREENTM cell lines, quality is front and center. We ensure the quality of our CRISPR gene-edited stable cell lines by focusing on:
1. Cell stability by single-cell cloning
2. Assay performance by directly screen in functional assays
3. Extensive quality control by expression and function after cell banking
To start the process, we collaborate with leading experts in CRISPR nuclease technologies enabling optimum gRNA design and optimization. In parallel, we determine the disease-biology relevant cell line and signaling pathway or phenotype for initial assay development. The parental cell line could be derived from specific tumors such as the lung, pancreas, liver, kidney, ovary, breast, nerve, bone etc. The assay could be based on signaling 2nd messenger, reporter, phenotype or binding in fluorescence, TR-FRET, and chemiluminescence readouts. For G-protein Coupled Receptors (GPCRs), for example, we have developed a wide spectrum of cell-based assays including Ca++, cAMP, β-arrestin, pGRK2, IP-1, pERK, pNFκB, radioligand binding, GTPγS, chemotaxis, insulin secretion, cell proliferation, luciferase reporter and internalization.
After transfection of CRISPR-gRNA, the stable pool of cells can be available for testing within 2 weeks. However, due to the heterogeneity of these cells, these stable pools are not amenable for drug discovery use where data consistency over months and years are critical. To tackle the stability issue, the Multispan team leverages over 100 years combined experience in cell sciences and cell signaling, to clone out cell lines that have proven to be stable for decades when properly stored.
By functional assay directly! Based on functional efficacy, heterozygous or homozygous stable cell lines can be detected and selected at the same time. The change of expression of the targets in the engineered clones will be further validated by flow-cytometry, western blotting, or RT-PCR. We have a 100% success rate for the generation of quality clonal stable cell lines for >500 targets. Furthermore, these highly characterized stable cell lines can be seamlessly integrated into our scaleup cell banking and compound screening services, which makes fast compound screening data turnaround possible.
