NEW YORK (GenomeWeb) – Scientists from Synthetic Genomics and the J. Craig Venter Institute have created a yeast-based platform for editing bacterial genomes using CRISPR/Cas9.
In a study published today in Scientific Reports, a team led by Venter and Daniel Gibson described how they engineered the small ribosomal subunit (16s) RNA of Mycoplasma mycoides, an essential gene for cell viability.
"In bacteria, rational mutagenesis of essential genes, often involved in fundamental biological process, is seldom accomplished in an efficient manner directly on the chromosome," the authors wrote. But using their genome-editing platform, they were able to get "robust and extensive site-directed mutagenesis directly on the bacterial chromosome with up to 100 percent efficiency," they said.
First, they cloned the entire synthetic bacterial genome into yeast artificial chromosomes, which had the ability to express Cas9. Then they edited the cloned genomes inside the yeast cell using CRISPR/Cas9 to snip at either end of the 16S rRNA gene, engaging the homologous recombination pathway to introduce donor templates for engineered genes. The new 16S rRNA genes featured genetic elements and helix substitutions from distantly-related bacteria, intended to interrogate the role of gene subunits.
After transplanting the genomes back into M. mycoides, the researchers studied how these changes affected cell viability. The output was simple: if the edit worked, the cells lived; if it didn't, they died.
"This work highlights the power of combining advanced genome editing and synthetic DNA technologies to build novel cells with unique characteristics," Gibson said in a statement.
In the narrow application of 16S rRNA editing, the platform enables scientists to break down the subunit function to better understand translation. Ribosomal RNA genes are particularly challenging to engineer, they said, because most genomes contain multiple copies.
In its work building minimum viable synthetic genomes, the Venter Institute has found a viable M. mycoides with only one copy of the gene. This is what they used to experiment with.
In many cases, editing the 16S rRNA gene crippled the cells and made them unviable. They went as far as making a version of the genome that lacked the gene altogether, which they said is not possible by conventional techniques.
But they were also able to restore the viability of that 16S rRNA-less genome by engineering it with wild-type or other engineered versions.
The authors suggested their process would help scientists interrogate essential genes and could advance the field of synthetic genomes.
"This new genomic platform would allow us to quickly engineer any essential gene in the 'simplest' M. mycoides genome and obtain a quick, binary 'yes' or "no" answer as to whether the modification introduced could support cellular viability," Krishna Kannan, an author on the paper, said in a statement.