Presenter: Silvana Konermann, Graduate Student in Dr Feng Zhang’s Lab at Massachusetts Institute of Technology
Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. The ease and scalability of the CRISPR-Cas9 system potentially enables systematic, genome-scale perturbation, but the magnitude of transcriptional up-regulation achieved by the first generation of Cas9 transcriptional activators typically ranges from low to ineffective.
In order to achieve a robust activator system, we undertook structure-guided engineering to generate a potent, synergistic Cas9 activation complex (SAM) capable of mediating robust up-regulation with a single sgRNA (Konermann et al., Nature 2014). SAM outperforms previous systems by more than two orders of magnitude. We further demonstrated that this system is capable of activating up to 10 genes simultaneously, allowing for understanding of complex genetic and regulatory networks.
Genome-scale gain-of-function screening approaches have largely been limited to the use of cDNA library systems, which are costly. To overcome this limitation, we designed a genome-scale sgRNA SAM library targeting every coding isoform from the RefSeq database (23,430 isoforms). We validated this screening approach by successfully identifying gain-of-function changes that can lead to the development of BRAF inhibitor resistance in BRAFV600 mutant melanoma cells. SAM activators present a highly reliable and generalizable tool for genome-wide interrogation of gene function and interaction in diverse biological processes.