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

ABSTRACT: To address a wide range of genetic diseases, genome editing tools that can achieve targeted delivery of large genes without causing double-strand breaks (DSBs) or requiring DNA templates are necessary. Here, we introduce 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 LINE-1 (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 this system, we demonstrate programmable insertion of a 1.1 kb gene expression cassette into specific genomic loci of human cell lines and primary T cells. Mechanistic studies reveal that CREATE editing 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 with broad therapeutic potential.

SUBMITTER: Yuxiao Wang

PROVIDER: S-SCDT-10_1038-S44319-024-00364-7 | biostudies-other |

REPOSITORIES: biostudies-other

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