Project Summary Genome editing technologies, such as CRISPR/Cas9 provide a rapid, and targeted means of both knocking out gene expression and knocking in gene modifications. Since our initial Phase I submission, the utility of CRISPR technology has expanded beyond the generation of cell lines, to forward genetic screening, in vivo manipulation of gene expression and even human therapeutics. Phase I efforts successfully demonstrated the use of the CellRaft Technology in a streamlined workflow for CRISPR-mediated genome editing in cell lines. Our Phase I report demonstrates several unique capabilities of the CellRaft technology for establishing genome edited cell lines using CRISPR: 1) performing all workflow steps (transfection, sorting, colony growth) on a single cell culture consumable; 2) releasing colonies from the array without disturbing the growth of other colonies (i.e. individual colony isolation, as opposed to en masse colony collection via trypsin); 3) sorting cells and colonies via imaging without requiring flow-based sorting methods which can damage cell health and perturb native phenotypes. By fully integrating the CRISPR workflow on a single platform, the CellRaft Array and the automated CellRaft AIR? System, the genome editing process will be dramatically streamlined. During Phase II we will continue validating this workflow and prepare for commercialization on the broader genome editing market. We will both scale up manufacturing of high-throughput CellRaft Arrays tailored to CRISPR/Cas9 genome editing under multiple conditions at once, as well as validate the performance of the system at two external laboratories. A new software package is also proposed which enables investigators to track transfection positive cells during their initial clonal colony growth phase. This software platform will also allow users to track the growth of colonies emerging from transfection positive single cells and sort them based on temporal propagation characteristics. Subawardee William Marzluff, PhD of UNC-Chapel Hill will evaluate the new high-throughput CellRaft Arrays as well as the new colony tracking software package and evaluate the AIR? System has a multi-lab core facility instrument. In a second subaward program, Mike McConnell, PhD of the University of Virginia will perform a time-course CRISPR experiment using the CellRaft Technology and automated AIR? System. Using both CRISPR-mediated genome editing and time-course tracking of colony growth, he will develop an in vitro model of tuberous sclerosis by editing the TSC1 gene in human IPSCs. Based on discussions with several CellRaft customers who use the system for CRISPR-based assays, there remains a clearly unmet need for a platform which broadly supports CRISPR genome editing workflows. The CellRaft Technology?s combination of imaging capabilities, support for the culture of viable cells and colonies and ability to sort and isolate cells for molecular analysis, lends itself to becoming a sufficiently flexible platform to enable a broad range of CRISPR-based experiments.