Leveraging Natural Systems for Large Genomic Insertions in Mammalian Cells

Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR–Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We discuss how systems such as PASTE can be generated, engineered for higher efficiency, and applied to practical therapeutic applications, especially treatment of monogenic disorders.

Learning Objectives:

1. Understand the current capabilities of DNA-editing modalities, including CRISPR-cas9, Prime editing, and base editors.

2. Discuss the strategies for tool development as it relates to PASTE and similar large genome insertion tools.

3. Develop knowledge on potential applications of large-genomic insertions and the potential drawbacks or advantages when compared to the current state of the art.