Unmet NeedTargeted genome editing has become a very important genetic tool with broad applications across various areas of science including but not limited to basic science, the pharmaceutical industry, and translational medicine. Presently, one of the most commonly used site-specific, genome editing systems is CRISPR/Cas9, as it enables insertion and deletion of specific gene fragments both
in vivo and
in vitro. However, CRISP/Cas9 along with other genome editing technologies only allow for editing on a per-gene basis and must be laboriously repeated for each gene of interest in a given system, limiting the throughput of the assay. Consequently, a protocol that allows for more efficient, multiplexing of template-based genome editing to target multiple genes at one time in a high-throughput manner is greatly needed.
Technology OverviewJohns Hopkins researchers have developed a set of scalable protocols that enable simultaneous generation of multiple edited cell lines in a high-throughput manner. The protocols have three main components. The first includes generating a library encoding sequences specific to the targeted genomic sites and in association with their corresponding DNA repair templates that carry the desired edits. The library design comprises constant elements consisting of needed machinery and variable parts with the gene/locus specific guide RNAs. The second element includes packaging and delivering the library with viral vectors expressing the needed genome editing components. Finally, the protocols include a specific methodology for isolating and conducting quality control of clonal lines that contain the desired transgenes.
Stage of DevelopmentThe inventors have generated a potential, scalable method for multiplexed genome editing based on the CRISPR/Cas9 system. Further optimization and research are required to test this method in various
in vivo and
in vitro mammalian systems across the numerous applications of targeted genome editing, including the addition of gene tags or replacing mutated genes.
