Modular safe-harbor transgene insertion for targeted single-copy and extrachromosomal array integration in...

Area of Science:

• Genetics
• Molecular Biology
• Developmental Biology

Background:

• Efficient transgenesis is crucial for genetic model organisms like C. elegans.
• Current methods can be limited in efficiency and reproducibility for targeted transgene insertion.

Purpose of the Study:

• To develop a modular safe-harbor transgene insertion (MosTI) system for C. elegans.
• To improve targeted insertion of single-copy transgenes and extrachromosomal arrays.
• To facilitate reproducible transgene expression in somatic and germ cells.

Main Methods:

• CRISPR/Cas9-mediated chromosomal integration using homology-directed repair or nonhomologous end-joining.
• Utilized split markers (unc-119, hygroR, gfp) for positive selection of integrated transgenes.
• Employed universal targeting vectors with common promoters and fluorophores.
• Leveraged short-read and long-read Nanopore sequencing for array characterization.

Main Results:

• MosTI achieved efficient single-copy transgene insertion (25-75%) using constitutive or inducible Cas9.
• Extrachromosomal array integration was also efficient (7-44%) at safe-harbor or endogenous loci.
• Reproducible transgene expression was observed in somatic and germ cells.
• Characterization of integrated arrays confirmed structure and copy number.

Conclusions:

• The MosTI system provides a versatile and efficient platform for transgene integration in C. elegans.
• Standardized vectors and protocols facilitate the construction of complex transgenic strains.
• This system will accelerate research into complex biological problems in C. elegans.