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Published on: April 30, 2018
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 06511, United States.
G3 (Bethesda, Md.)
|May 28, 2026
Summary
FLInt 2.0 enhances transgene integration in C. elegans by reducing false positives. This refined CRISPR-Cas9 method improves screening efficiency for generating stable transgenic lines.
Area of Science:
- Genetics
- Molecular Biology
- Developmental Biology
Background:
- Transgenesis in *C. elegans* allows precise gene expression control.
- Fluorescent Landmark Interference (FLInt) facilitates transgene integration via CRISPR-Cas9.
- FLInt 1.0 suffered from high false-positive rates, complicating screening.
Purpose of the Study:
- To develop an improved FLInt strategy (FLInt 2.0) to minimize false positives in transgene integration.
- To enhance the efficiency and reliability of generating stable transgenic *C. elegans* lines.
Main Methods:
- FLInt 2.0 utilizes targeted CRISPR-Cas9 cutting of fluorescent protein landing sites.
- Non-fluorescent F2 animals are used to identify stable, multi-copy transgenic lines.
- Investigated the impact of DNA structure (linear vs. circular) on integration efficiency.
Main Results:
- FLInt 2.0 significantly reduces false positives compared to FLInt 1.0.
- Non-fluorescent F2 generation reliably indicates successful transgene integration.
- Linear DNA substrates enhance transgene array formation and insertion efficiency.
- Subsequent Cas9 targeting allows fine-tuning of expression levels in multicopy lines.
Conclusions:
- FLInt 2.0 offers a robust, visually guided method for site-specific transgene integration in *C. elegans*.
- This approach streamlines the generation of transgenic lines, increasing experimental throughput.
- The strategy provides a generalizable framework for improving transgene integration techniques.
