(W1191) Rapid, easy, and efficient biallelic knock-in system unified with an independent second synthetic cistron for personalized transgene expression
PROJECT SCIENTIST CEDARS-SINAI MEDICAL CENTER, California, United States
Abstract: Unspecific, inefficient, lack of control of the copy number and/or small cargo knock-in of transgenic elements confines the capacity of cell and gene therapies. For example, viruses and transposases allow quick integration of considerable cargo sizes into the genome but lack both specificity and controlled copy number. Nucleases like TALENS and CRISPR-associated proteins help integrate large cargo sizes into specific loci, but they rely on homology-directed repair, which is very inefficient. Thus, the selection of the edited cells is laborious and time-consuming. Prime editing is another system that allows small editions into the targeted loci and combined with recombinases it can be used to insert big fragments, however, the selection of monoallelic or biallelic edited cells is still an arduous task. To address these challenges, we have developed a highly efficient methodology for inserting large cargo into a specific locus while enabling the rapid selection of biallelic knock-in cells. Furthermore, transgenic elements can be expressed under any synthetic promoter, making the system very customizable. In human cells, we achieve this by targeting a constitutive gene with template vectors and ribonucleoprotein complexes to insert the desired landing pad via homology-directed repair. The landing pad enables both the visualization and selection of biallelic-edited cells while also supporting the addition of a secondary cistron, where new transgenic elements can be expressed under any synthetic promoter and inserted via recombinases. As proof of principle, we tested our system using fluorescent gene reporters for easy visualization and selection of cells. However, any transgene can be inserted, making the system adaptable for testing therapeutic genes or mutated genes for disease modeling under the expression of synthetic promoters.
