A CRISPR/CAS9 BASED ENGINEERING TOOL TO ACTIVATE EXPRESSION OF MULTIPLE GEN INDIVIDUALLY OR IN ANY SPECIFIC COMBINATION

摘要

Engineering of cells by overexpression or knock-down/out of individual genes has demonstrated that in most cases the manipulation of single genes is not sufficient to alter a cellular phenotype. Rather, multiple genes involved in a pathway need to be manipulated. Especially in mammalian cells such as CHO, where clonal variation is large, it has been difficult to unequivocally assess whether the observed change in phenotype is due to such clonal variation or the engineered gene. This can in part be overcome by testing multiple subclones, however, once it comes to engineering multiple genes and combinations thereof, the required workload quickly becomes prohibitive. We here present a simple technology for successive and/or specific activation of multiple genes integrated into a single genomic locus, which presents a potential solution to this problem.