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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 06511, USA.

G3 (Bethesda, Md.)

|May 28, 2026

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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.

Keywords:

C. elegans CRISPR WormBase multiplex gene expression safe landing sites transgenesis

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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.