Endogenous Tagging Strategies for Accelerated Target Validation and Detection

CRISPR/Cas9 system has facilitated precise insertions in the genome of a cell to monitor endogenous activities of genes. This method has advantages compared to target overexpression under an exogenous promoter which may cause numerous artifacts including ectopic sub-cellular localizations and erroneous formation of protein complexes. As opposite to an irreversible gene knockout, precise temporal and spatial control of protein expression enables a detailed analysis of the cellular phenotype or the investigation of essential genes. PROTACs (Proteolysis-Targeting Chimera) may be combined with CRISPR to establish a universal approach to generate cell models for Target Validation (TV) where the target is fused to both a tag sensitive to PROTAC degradation (such as the HaloTag), and a split nanoluciferase.

We have developed a rapid workflow for cell line engineering based on an endogenous tagging strategy utilizing long single-stranded DNA (ssDNA; 2000-4000 nt in length) HDR donors. Knockin pools or clones can then be used in protein degradation assays via PROTAC to interrogate the protein function. The decrease of the target protein and the speed of the protein degradation can be readily monitored by luciferase activity.