Improving Transgenesis Efficiency and CRISPR-Associated Tools Through Codon Optimization and Native Intron Addition...

Area of Science:

• Molecular Biology
• Genomics
• Nematology

Background:

• Molecular tools are crucial for mechanistic studies but limited in many organisms.
• Transgenesis and CRISPR-engineering applications are restricted, especially within the phylum Nematoda.
• Low transgenesis efficiency in non-model nematodes may stem from transgene incompatibilities.

Purpose of the Study:

• To investigate genomic features influencing transgenesis across diverse nematode species.
• To enhance transgenesis efficiency in nematodes through molecular tool optimization.
• To develop improved tools for genetic manipulation in non-model nematodes.

Main Methods:

• Comparative genomic analysis of codon usage bias and intron composition in 10 nematode species.
• Codon optimization and native intron addition for transgenes in Pristionchus pacificus.
• Development of a fluorescent co-injection marker for CRISPR-edited individuals.

Main Results:

• Significant differences in codon usage bias and intron composition were observed across the studied nematode species.
• Transgenesis efficiency in Pristionchus pacificus was substantially improved using optimized transgenes.
• A novel fluorescent marker facilitated easier and faster detection of CRISPR-edited P. pacificus.

Conclusions:

• Genomic feature analysis provides insights into nematode transgenesis barriers.
• Codon optimization and intron manipulation are effective strategies to enhance transgenesis in nematodes.
• Improved molecular tools, including fluorescent markers, accelerate genetic research in non-model organisms.