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

Area of Science:

• * Molecular Biology
• * Genetics
• * Developmental Biology

Background:

• * Caenorhabditis 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, like the Self-Excising Cassette (SEC), have limitations for essential genes.

Purpose of the Study:

• * To develop an improved method for generating knock-in alleles in C. elegans, particularly for essential genes.
• * To overcome the loss-of-function issue associated with SEC before marker excision.
• * To enable the isolation of homozygous lines for essential genes prior to selectable marker removal.

Main Methods:

• * Development of a Nested, Self-Excising selection Cassette (NSEC) integrated into a synthetic intron.
• * NSEC is designed to not interfere with endogenous NSEC-fusion protein expression.
• * NSEC incorporates options for fluorescent proteins (e.g., GFP, mScarlet-I) and an auxin-inducible degron tag.

Main Results:

• * NSEC allows for the isolation of homozygous lines for N-terminally and internally tagged essential genes before cassette excision.
• * Endogenous protein function is preserved throughout the process.
• * The method provides a standardized workflow for generating knock-ins in any C. elegans background without genetic balancers.

Conclusions:

• * The NSEC system significantly enhances the efficiency, scalability, and versatility of C. elegans genome engineering.
• * This tool simplifies the generation of knock-in strains, expanding research possibilities.
• * NSEC facilitates the study of essential genes through seamless tagging and functional analysis.