PROJECT SUMMARY Genome editing is revolutionizing biomedicine and biotechnology by enabling the precise modification of genomic DNA in living cells. While various genome-editing tools have been developed over the past decade, the CRISPR-Cas9 system has emerged as a particularly versatile and efficient technology for editing DNA. Nonetheless, limitations derived from its reliance on DNA double strand breaks (DSB), which can lead to unpredictable editing outcomes and even chromosomal translocations, could limit its applications. Base editors (BEs) and prime editors (PEs) are two novel classes of genome-editing tools capable of introducing precise single-base conversion in DNA without the requirement of a DSB. PEs, in particular, provide greater flexibility than BEs, owing to their ability to introduce any type of base conversion and even programable small insertions and deletions. This expanded set of capabilities compared to other technologies makes PEs a particularly promising platform for applications in biomedicine; however, the large size of PEs precludes their in vivo delivery by AAV, a promising and effective gene delivery vehicle that is currently under evaluation in multiple clinical trials. To overcome these obstacles, we have created a split-PE platform that is compatible with AAV delivery and have demonstrated the functionality of this approach in cultured cells. Despite this progress, there still remain several critical challenges, which we here propose to overcome in order to optimize this technology for effective and specific in vivo prime editing. To accomplish this objective, we have assembled a multidisciplinary team with collective expertise in genome editing (Dr. Perez-Pinera), AAV gene delivery (Dr. Gaj) and computational biology (Dr. Song). Our collaborative efforts will yield an integrated and comprehensive PE toolset that will blend strategies for target identification and editing optimization, with methods for reducing off-target effects and immune responses, thus priming this technology for future in vivo applications. Given that the flexibility of PEs has significantly expanded the number of actionable target sites that can be genetically modified, we anticipate that the integrated technologies we develop will have large, direct and long- lasting impact in biomedicine by enabling not only novel gene therapies, but also basic research. In particular, our technology will provide investigators with biological tools that are uniquely capable of introducing mutations within post-mitotic cells in vivo, which could be used to dissect functional elements or even determine the role of pathogenic mutations in a cell- and tissue-specific manner. The technologies created by this application will thus broadly impact biotechnology and biomedicine.