Project Summary/Abstract (max: 30 line limit) Adult neurons in the central nervous system fail to regenerate after spinal cord injury (SCI). Large-scale screens performed in vitro and in vivo have identified many genes that regulate axonal regeneration, including PTEN, SOCS3, c-Myc, the KLF family of transcription factors, and more. In the optic nerve model, the greatest enhancement of axonal regeneration is achieved by targeting multiple genes in combination, such as co-deletion of PTEN and SOCS3, or PTEN deletion combined with overexpression of c-Myc and CNTF. However, no screens have systematically compared the combined knockdown or overexpression of different gene combinations, and the power of large in vivo screens may be limited by the need to individually test every candidate and between-subject variability. In this study, we propose to perform the first pooled in vivo screening of regenerative combination therapies in a mouse model of SCI, which could identify new combinations of genetic targets that more potently enhance both the number of regenerating axons and the distance of axonal regeneration after SCI. We will employ newly available tools for genetic knockout and overexpression to efficiently perform multiple in vivo screens, using a Cre-dependent barcode and next-generation DNA sequencing to simultaneously compare a pooled library of 36 candidates in a single animal. Our proposal is divided into two Aims. Aim 1 will use CRISPR to first compare the genetic knockout of 36 individual candidate genes that are known to limit axonal regeneration. After identifying the 9 individual knockouts that most potently enhance regeneration, we will then screen every possible pair of the top 9 gene knockouts in combination. Aim 2 will first compare the overexpression of 36 individual candidate genes that are known to promote regeneration. We will then combine the 6 most effective overexpressed genes with the 6 most effective knockout pairs to screen 36 unique combinations of one overexpressed gene and two gene knockouts. Every screen will use a pooled library to directly compare all 36 candidates within the same animal. To our knowledge, systematic screening of multiple gene knockouts, or of combined knockout and overexpression, has not been previously performed in any model of axonal regeneration. Future studies will evaluate the functional benefit of the most effective combination therapies in a clinically relevant SCI model. We thus propose a new method for pooled in vivo screening of genetic therapies for SCI, which could identify new translational combination therapies that enhance axonal regeneration and improve functional recovery.