FLInt 2.0: Robust and customizable single shot integration in <i>C. elegans</i>

Transgenesis in <i>Caenorhabditis elegans</i> has revolutionized biological research by enabling the precise control of expression of both endogenous and exogenous genes. The FLInt (Fluorescent Landmark Interference) method was developed to accelerate site-specific integration of transgenes, but persistent false-positive events during screening have emerged as a rate-limiting step. Here, we present an alternative FLInt strategy, FLInt 2.0, that reduces false positives by biasing Cas9 cutting of the tdTomato and Cbr <i>unc-119</i>(+) safe-harbor locus, adjacent to the untranslated region (3' UTR). The design preserves fluorescence during non-integrative repair events, such that only true integration of an extrachromosomal array abolishes tdTomato expression. We demonstrate that this targeted approach maintains high integration efficiency while significantly decreasing the proportion false positives observed in F1 progeny. Molecular and transmission analyses confirm that non-fluorescent F2 animals reliably represent stably integrated multi-copy transgenic lines, which can be tailored to desired expression levels using a simple subsequent Cas9 targeting approach. By exploiting coding-frame geometry to discriminate integration events from repair-induced artifacts, this method streamlines the identification of the true integrants, reducing labor-intensive screening and increasing experimental throughput. Our strategy provides a robust, visually guided, and efficient refinement of FLInt, offering a generalizable framework for improving site-specific transgene integration in <i>C. elegans</i>.