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

Abstract Transgenesis in Caenorhabditis elegans has revolutionized biological research by enabling the precise control of expression of both endogenous and exogenous genes. FLInt (Fluorescent Landmark Interference) was developed to integrate transgenes via CRISPR-Cas9 using visible changes in existing fluorescent protein expression strains. While the original FLInt method (FLInt 1.0) enabled a simple visual readout of potential transgene integration, the process was prone to false positives, leading to burdensome screening efforts. Here, we present an alternative FLInt strategy, FLInt 2.0, that reduces false positives by targeted CRISPR-Cas9 cutting of fluorescent protein landing sites in a manner which largely retains fluorescence in non-integrative repair events but eliminates expression upon transgene integration. We demonstrate that this targeted approach maintains effective integration while significantly decreasing the proportion of false positives. Molecular and transmission analyses confirm that non-fluorescent F2 animals more reliably represent stably integrated multi-copy transgenic lines. We show that integration efficiency and array transmission are influenced by DNA structure and composition, with linear DNA substrates promoting more robust array formation and insertion. We further show that multicopy transgene lines can be tailored to desired expression levels using a simple subsequent Cas9 targeting approach, reducing labor-intensive screening and increasing experimental throughput. Our strategy provides a robust, visually guided refinement of FLInt, offering a generalizable framework for improving site-specific transgene integration in C. elegans.