CRISPR-Enabled Autonomous Transposable Element (CREATE) for RNA-based gene editing and delivery

To address a wide range of genetic diseases, genome editing tools that can achieve targeted delivery of large genes without causing double-stand breaks (DSBs) or requiring DNA templates are necessary. Here, we introduce the CRISPR-Enabled Autonomous Transposable Element (CREATE), a genome editing system that combines the programmability and precision of CRISPR/Cas9 with the RNA-mediated gene insertion capabilities of the human LINE1 (L1) element. CREATE employs a modified L1 mRNA to carry a payload gene, and a Cas9 nickase to facilitate targeted editing by L1-mediated reverse transcription and integration without relying on DSBs or DNA templates. Using the system, a 1.1 kb gene cassette comprising an EF1α promoter and green fluorescent protein (GFP) gene is inserted into several genomic loci of multiple human cell lines. Mechanistic studies reveal that the CREATE system is highly specific with no observed off-target events. Together, these findings establish CREATE as a programmable gene delivery technology solely based on RNA components, enabling large-scale in vivo genome engineering with broad therapeutic potential. ### Competing Interest Statement All authors are/were employees of MyeloidTx and hold equity interest in MyeloidTx. Sequences including primers, sgRNAs are available in the supplementary materials.