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Nearly complete redirection of insertion-type indel into recombination enhances knock-in and facilitates endogenous biomolecular condensate analysis

ABSTRACT: Biomolecular condensate is crucial for cellular organization and function, yet its study faces significant technical challenges1,2. Cells often operate near a phase separation threshold, where even slight overexpression can profoundly alter condensate behavior3. Despite this sensitivity, many studies continue to rely on ectopic expression for in vivo measurements4. This reliance underscores the limitations of current knock-in (KI) techniques, where the inefficiency of homologous directed repair (HDR) is often outcompeted by the error-prone non-homologous end joining (NHEJ) pathway, frequently resulting in unwanted insertions and deletions (indels) rather than KI5. Newer base editing (BE) and prime editing (PE) technologies, though more precise, remain constrained5. BE only performs specific base substitutions, and PE is limited to short edits near PAM sequences. Neither can efficiently KI complex sequences needed for studying condensates. For example, modifying polyQ tract of ATXN2, a long repetitive sequence linked to neurodegeneration6,7, remains challenging. Besides, unintended off-target effects and chromosomal rearrangements have raised concerns about the safety of KI technology in practical applications. Therefore, a highly efficient and versatile KI technology is needed as a reliable tool for studying the intricate dynamics of biomolecular condensates.

ORGANISM(S): Homo Sapiens

SUBMITTER: Wei Qin

PROVIDER: PXD068953 | iProX | Sat Sep 27 00:00:00 BST 2025

REPOSITORIES: iProX

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