Project Abstract In this Administrative Supplement grant application, we request funds to purchase a new cell sorter (S3e™ Cell Sorter, Bio-Rad). This state of art cell sorter will enable us to evaluate large size gene knock-in events, including both on-target knock-in, on-target insertion/deletion (indel) rates as well as potential off-target knock-in and indel events, at the single cell resolution. In Aim 1, we plan to use this cell sorter to evaluate mi-spCas9-v2. In Aim 2, we will use this cell sorter to evalute mi-saCas9. In Aim 3, we will use this cell sorter to evaluate the efficacy and safety profiles of miCas9 in clinically relevant applications. As a results, this new cell sorter, once equipped, will generate more convincing data to demonstrate the advantages of the proposed miCas9 gene editing nucleases.