Dr. J Wickenden, Team Leader, Horizon Discovery
The CRISPR–Cas9 system can be used to disrupt specific genes leading to genetic knockouts. Thus, it has an advantage over traditional RNAi-based target validation assays in which incomplete knockdown can lead to ambiguous results. However, use of the CRISPR–Cas9 system has revealed that it can result in insertions and deletions (indels) that are in frame and transcription of the gene is maintained. Such cases enable cells which are dependent on the target gene to carry on proliferating, falsely indicating that the gene knockout can be tolerated. To overcome this, we have developed the Pathfinder Target Essentiality Assay, a medium-throughput assay to analyze CRISPR–Cas9-mediated gene editing on a clonal level to verify the zygosity and therefore the essentiality of the targeted gene.