One step engineering of the small-subunit ribosomal RNA using CRISPR/Cas9

Area of Science:

• Molecular Biology
• Microbial Genomics
• Synthetic Biology

Background:

• Bacteria are crucial models for molecular biology due to simple genomes.
• Engineering bacterial chromosomes is key to understanding gene function.

Purpose of the Study:

• To demonstrate the engineering of the small-ribosomal subunit (16S) RNA in Mycoplasma mycoides.
• To investigate the functional resilience of engineered 16S rRNA genes.

Main Methods:

• Cloning the M. mycoides genome in yeast.
• Utilizing CRISPR/Cas9 and yeast recombination for 16S rRNA gene engineering.
• Introducing engineered genes via genome transplantation.

Main Results:

• Successfully engineered 16S rRNA genes in M. mycoides.
• Observed surprising resilience of the engineered 16S rRNA gene to genetic additions and helix substitutions.
• Demonstrated functional viability through genome transplantation.

Conclusions:

• The developed system enables functional studies of 16S rRNA.
• The engineered M. mycoides genome can serve as a platform for studying other genetic elements and fundamental life processes.