Programmable large-cargo integration: Overcoming size constraints for next-generation gene therapy

The emergence of base and prime editors-genome editing tools that avoid double-strand breaks (DSBs)-has enabled precise point mutations, insertions, inversions, deletions, and substitutions, which accelerates the development of single-intervention therapies and advances individualized genomic medicine. However, their limited efficiency in inserting large DNA fragments has restricted applications for correcting diverse pathogenic mutations within a single gene. In this review, we explore three recently developed strategies for efficient large DNA cargo insertion (>1 kb): CRISPR-associated Tn7-like transposases (CASTs), PE-integrase systems, and R2 retrotransposon fusions (nCas9-R2). We examine the applications of these systems in both bacterial and mammalian contexts and discuss their respective advantages and current limitations. Finally, we address persistent challenges and propose potential directions to guide future research.