Crispr-Based Genome Editing Tools: New Applications and Streamlined Workflows

CRISPR-Cas9 systems provide a platform for high efficiency genome editing that can lead to innovative applications in cell engineering.  However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain two steps that limit overall efficiency and general ease of use.  Here we describe novel methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA complexes into a variety of cells through liposome-mediated transfection or electroporation. We will present a streamlined cell engineering workflow that goes from gRNA design to analysis of edited cells in as little as three days and results in highly efficient genome editing and biallelic knockout of multiple genes in hard-to-transfect cells.  The reagent preparation and delivery to cells requires no plasmid manipulation so is amenable for high throughput, multiplexed genome-wide cell engineering.

Further, we will show data using lentivirus-based CRISPR delivery for high-throughput screening of mammalian cell populations.  Until recently RNAi has been the main tool for performing loss of function studies in mammalian cell populations.  Using CRISPR technology, we are now able to perform complete gene knock-out studies which hold promise for clearer phenotypes and fewer false readouts as what is currently seen with the variable knock-down of expression using RNAi.  We are creating gene family-specific arrayed libraries of CRISPR-lenti particles.  These new tools enable high throughput, arrayed gene knockout screens using various cell types, including our proprietary CellSensor lines that give a fluorescent readout of various signaling pathway activities.

These two CRISPR-based gene-editing platforms represent the latest in the rapid evolution of editing tools for mammalian genomes by simplifying and increasing the cell engineering workflow and providing a pre-designed, ready to use platform for efficient compound screening in mammalian cell lines.