Engineering the C. elegans genome with a nested, self-excising selection cassette.

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• *C. elegans* is a key model organism for *in vivo* biological studies.
• Targeted knock-in alleles in *C. elegans* allow visualization and functional modification of endogenous proteins.
• Existing genome engineering methods, including the Self-Excising Cassette (SEC), have limitations for essential genes.

Purpose of the Study:

• To develop a simplified method for generating knock-in alleles of essential genes in *C. elegans*.
• To overcome the loss-of-function issue associated with traditional SEC methods for essential genes.
• To enhance the efficiency and versatility of *C. elegans* genome engineering.

Main Methods:

• Development of a Nested, Self-Excising selection Cassette (NSEC) system.
• Integration of NSEC within a synthetic intron to avoid interfering with gene expression.
• Design of NSEC variants with optional auxin-inducible degron and various fluorescent proteins (mTurquoise2, GFP, mStayGold, mNeonGreen, mScarlet-I).

Main Results:

• The NSEC system allows for the isolation of homozygous lines for N-terminally tagged essential genes prior to selectable marker excision.
• NSEC does not interfere with the expression of endogenous N-terminally tagged fusion proteins.
• The NSEC system provides a standardized workflow for generating N-terminal and internal tags in any genetic background without requiring genetic balancers.

Conclusions:

• The NSEC system significantly simplifies the generation of knock-in alleles for essential genes in *C. elegans*.
• This innovation expands the molecular toolbox for *C. elegans* researchers.
• NSEC enhances the scalability, efficiency, and versatility of genome engineering in *C. elegans*.