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FLInt 2.0: Robust and customizable single shot integration in C. elegans
Nawaphat Malaiwong1, Porhathai Malaiwong1, Chloe Kim1
1Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA.
Biorxiv : the Preprint Server for Biology
|February 6, 2026
Summary
FLInt 2.0 reduces false positives in Caenorhabditis elegans transgenesis by biasing Cas9 cutting. This refined fluorescent landmark interference method improves screening efficiency for stable transgenic lines.
Area of Science:
- Molecular Biology
- Genetics
- Developmental Biology
Background:
- Transgenesis in *Caenorhabditis elegans* allows precise gene expression control.
- The FLInt method accelerates transgene integration but suffers from false positives.
- Efficient site-specific integration is crucial for genetic research.
Purpose of the Study:
- To develop an improved FLInt strategy (FLInt 2.0) to reduce false positives in transgene integration screening.
- To maintain high integration efficiency while decreasing screening errors.
- To streamline the identification of true transgenic lines in *C. elegans*.
Main Methods:
- Developed FLInt 2.0 by biasing Cas9 cutting at the tdTomato and Cbr *unc-119* (+) safe-harbor locus.
- Utilized the preservation of tdTomato fluorescence during non-integrative repair events.
- Employed molecular and transmission analyses to confirm stable integration in F2 progeny.
Main Results:
- FLInt 2.0 significantly reduced false-positive events in F1 progeny.
- High transgene integration efficiency was maintained.
- Non-fluorescent F2 animals reliably indicated stably integrated multi-copy transgenic lines.
Conclusions:
- FLInt 2.0 offers a robust and visually guided refinement of the FLInt method.
- The strategy enhances experimental throughput by reducing labor-intensive screening.
- This approach provides a generalizable framework for improving site-specific transgene integration in *C. elegans*.
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