Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Genome engineering aims to streamline custom DNA modifications.
• CRISPR/Cas9 technology offers powerful gene editing capabilities.
• Efficient generation of knock-in alleles is crucial for functional genomics.

Purpose of the Study:

• To present a novel selection strategy for fluorescent protein (FP) knock-ins using CRISPR/Cas9-triggered homologous recombination.
• To reduce the time and labor involved in generating custom genome modifications in Caenorhabditis elegans.
• To develop a method for generating FP fusions, transcriptional reporters, and loss-of-function alleles in a single step.

Main Methods:

• A self-excising cassette (SEC) containing a drug-resistance gene, phenotypic marker, and inducible Cre recombinase was designed.
• The FP-SEC module, flanked by LoxP sites within a synthetic intron, was inserted into C. elegans genes.
• CRISPR/Cas9-triggered homologous recombination was used for knock-in.
• Heat shock induced Cre recombinase activity, leading to SEC excision.

Main Results:

• The FP-SEC module was successfully integrated into C. elegans genes.
• SEC self-excised upon heat shock, leaving only the FP tag.
• The method eliminated the need for PCR screening and a second injection step for marker removal.
• This approach enabled the generation of FP fusions, transcriptional reporters, and loss-of-function alleles efficiently.

Conclusions:

• This novel selection strategy significantly simplifies and accelerates the generation of fluorescent protein knock-ins in C. elegans.
• The self-excising cassette (SEC) system minimizes hands-on labor and exogenous sequences.
• The method provides a versatile tool for creating various genetic modifications in a single step, advancing genome engineering applications.