The scientists at J. Craig Venter Institute (JCVI) and Synthetic Genomics Inc (SGI) continue their quest to better understand life by creating it. Following the team’s milestone of creating the smallest genome in an independent organism this past March, JCVI/SGI have now published a one-step process for engineering Mycoplasma mycoides 16s rRNA using a combination of CRISPR/Cas9 gene editing technology and yeast recombination machinery.
The JCVI/SGI team is led by Krishna Kannan, Ph.D., SGI and senior author Daniel G. Gibson, Ph.D., SGI and JCVI, who have highlighted the system’s importance not only in the study of the function of 16S rRNA, but also as a way of converting M. mycoides genome into a platform to study other genetic structures and thus answer fundamental questions of life.
This development could provide an efficient, high throughput tool to test engineered essential genes, such as the 16S rRNA. “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” said Kannan through a recent press release. “Using this platform, we observed a surprising resilience of the 16S rRNA gene when extensive modifications were introduced”.
rRNAs had previously presented a challenge for synthetic biologists because of their presence as multiple copies in almost every genome. This method could now accelerate the study of these structures as the most conserved genes in all branches of life, giving new insights to evolutionary history.
The combination of CRISPR/Cas9 and yeast homologous recombination machinery was achieved by cloning the M. mycoides genome in a strain of yeast expressing Cas9 and then converting it to a non-functional form through the replacement of the “rrs” gene (which encodes for 16S rRNA) with a ura3 yeast marker. This ura3 gene was then replaced with synthetically engineered 16s rRNA cassettes by making use of in vitro transcribed guide RNAs. The resulting 16S rRNA cassettes were tested by transplanting them into M. capricolum recipient cells, where they were able to convert the genome to a functional state, thus confirming their life supporting capacities.
The JCVI/SGI partnership continues to be an exemplary case of synthetic biology pioneers. Since their first joint studies in 1999, the team has developed numerous genetic tools that have culminated in the creation of Mycoplasma mycoides JCVI-syn1.0 in 2010, advancing to the JCVI-syn3.0 version this March and presumably moving at an accelerated pace to a fourth, fifth and other successive versions as new genetic machineries are discovered, combined and upgraded.0