Nearly complete redirection of insertion-type indel enhances knock-in and facilitates endogenous biomolecular condensate analysis
Biomolecular condensates play critical roles in cellular organization and are implicated in many diseases. Because condensate properties are highly sensitive to concentration changes, gene knock-in (KI) is the preferred method for studying condensate-forming proteins in their natural context. However, the limitations of current gene editing tools make it difficult to fully utilize KI for condensate studies, while alternative methods often lead to artifacts in condensate studies. Here, we introduce RecT7-KI, a technology that addresses these challenges by bypassing endogenous checkpoints, significantly enhancing the efficiency of long and complex KIs. RecT7-KI enhances KI by converting insertion-type indels to recombination events. Even at loci with very high insertion rates, it can also redirect these outcomes, substantially increasing KI. In addition, RecT7-KI significantly reduces large deletions commonly seen with CRISPR-based editing. Using RecT7-KI, we performed KI analysis of clinical variants, heterozygous mutations, and polyQ repeat expansions in condensate-forming proteins. This enabled detailed studies of condensate processing, dynamics, and composition entirely through KI. The RecT7-KI system is efficient, versatile, and minimizes artifacts, making it a valuable tool for studying biomolecular condensates.