Establishing CRISPR mediated whole-genome knock-out screening to identify essential genes in nephrovascular development
A crucial pursuit of the Center of Excellence in Pediatric Nephrology is understanding how precursor cells differentiate and acquire their characteristic phenotypes. The transformation of one cell type into another requires the participation of multiple regulatory regions along the genome. Using CRISPR/Cas9 technology it may finally be possible to ascertain -at a genome-wide scale- the key genes involved in the fate of a cell.
CRISPR/Cas9, a bacterial immune response system against environmental viruses, has been adapted to function in mammalian cells utilizing the endonuclease (Cas9) and two short guide RNAs (sgRNA) for targeting a specific gene. CRISPR mediated targeted gene editing and regulation technology has generated tremendous excitement owing to its simplicity, efficiency and multiplex targeting functionality. However, like any other genome editing technology, potential off targeting effects of the system remain a major concern. This proposal builds upon our findings that off-targets are enriched in genes and promoters, potentially confounding experimental results obtained by CRISPR-mediated targeted gene editing. We plan to establish and further improve the CRISPR technology for whole genome knock-out (KO) screenings. Current whole genome targeting sgRNA libraries are composed of thousands of sgRNAs that target every single gene in the genome with an average of ~5 sgRNA/gene. To reduce potential off-targets of the system, we will improve the current mouse and human library contents so that each gene in the genome will be targeted by more than 10 unique sgRNAs to achieve CRISPR/Cas9 mediated genetic knock-outs. Initially, the technology will be tested using well-established protocols for cells already in our laboratory. Once the reliability and functionally of the technology has been validated, CRISPR-Cas libraries will be adapted for genome-wide identification of essential genes for specific cellular phenotypes to allow us to identify key regulators of nephron and vascular development.
We will perform these experiments in collaboration with members of the Center of Excellence in Pediatric Nephrology. Once established, the technology will be made available to the nephrology community at large.