Teams at Venter Institute and Synthetic Genomics, Inc. Successfully Engineer 16S rRNA using One Step Process Combining CRISPR/Cas9 Systems and Yeast Recombination Machinery

LA JOLLA, Calif., Aug. 4, 2016 /PRNewswire/ -- Researchers from the J. Craig Venter Institute (JCVI) and Synthetic Genomics, Inc. (SGI) have published research describing a method for engineering Mycoplasma mycoides 16S ribosomal RNA (rRNA) using a one-step process that combines CRISPR/Cas9 gene editing system with yeast recombination machinery.

The rRNAs are some of the most conserved genes in all branches of life and thus are used to trace evolutionary history. While they are fundamental to the process of protein synthesis, to date they have been challenging to engineer since they are present as multiple copies in almost every genome.

The JCVI/SGI team, led by Krishna Kannan, Ph.D., SGI and senior author Daniel G. Gibson, Ph.D., SGI and JCVI, believe that this new combined technique has wide applications in the field of synthetic biology and can be used to both study the function of 16S rRNA specifically but also to help more broadly interrogate any genetic structure and answer basic questions of biology.

Bacteria, such as E. coli, are widely used in research as model organisms to study basic biological functions because of their often small and simple genomes and gene architectures. While the development of CRISPR/Cas9 systems have made gene editing much easier in some prokaryotic organisms, their utility has still been generally limited in many bacteria. Given the JCVI/SGI team's expertise in development of many new tools in synthetic biology in their quest to construct the first synthetic bacterial cell, they set out to see if a combination of these tools and CRISPR/Cas9 could bring advances to the field.  

The team began the process to edit the M. mycoides genome by first cloning it in a strain of yeast expressing Cas9.  This genome was subsequently converted into a non-functional form through replacement of the essential "rrs" gene (that encodes for the 16S rRNA) with a ura3 yeast marker. By using in vitro transcribed guide RNAs, this ura3 gene was replaced with synthetically engineered 16S rRNA cassettes in yeast. The capacity of these16S rRNA cassettes to support life (by converting the genome to a functional state) was tested by genome transplantation from yeast into M. capricolum recipient cells.

By combining CRISPR/Cas9 editing technology with the yeast homologous recombination machinery, genome transplantation and other tools developed by the JCVI/SGI team, the group has developed an efficient, high throughput platform to test engineered essential genes like the 16S rRNA thus facilitating more experiments into biological function and helping to answer basic questions of life.

"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. Using this platform, we observed a surprising resilience of the 16S rRNA gene when extensive modifications were introduced," said Dr. Kannan, Scientist, Synthetic Systems and DNA Technologies Group, SGI.

"This work highlights the power of combining advanced genome editing and synthetic DNA technologies to build novel cells with unique characteristics," said Dr. Gibson, Vice President, DNA Technologies, SGI; Associate Professor, JCVI.

The paper describing this research is being published today in the journal Scientific Reports. Other JCVI and SGI researchers on this paper are: J. Craig Venter, Ph.D., Hamilton Smith, M.D., Clyde Hutchison, Ph.D., John Glass, Ph.D., Chuck Merryman, Ph.D., Billyana Tsvetanova, Ph.D., Ray-Yuan Chuang, Ph.D., Vladimir Noskov, Ph.D., Nacyra Assad-Garcia, and Li Ma.

This work was funded by SGI.

The JCVI/SGI team has a long and successful history in synthetic biology research. The team, who published some of their first studies as early as 1999, culminated their efforts with the first synthetic cell, Mycoplasma mycoides JCVI-syn1.0 in 2010, and in March 2016 published result of the successful construction of the first minimal synthetic cell, JCVI-syn3.0. This cell contains 531,560 base pairs and just 473 genes, making it the smallest genome of any organism that can be grown in laboratory media.

About J. Craig Venter Institute

The JCVI is a not-for-profit research institute in Rockville, MD and La Jolla, CA dedicated to the advancement of the science of genomics; the understanding of its implications for society; and communication of those results to the scientific community, the public, and policymakers. Founded by J. Craig Venter, Ph.D., the JCVI is home to approximately 200 scientists and staff with expertise in human and evolutionary biology, genetics, bioinformatics/informatics, information technology, high-throughput DNA sequencing, genomic and environmental policy research, and public education in science and science policy. The JCVI is a 501 (c)(3) organization. For additional information, please visit http://www.JCVI.org.

About Synthetic Genomics Inc.

Synthetic Genomics Inc. (SGI), located in La Jolla, CA, is a leader in the fields of synthetic biology and synthetic genomics, advancing genomics to better life. SGI applies its intellectual property in this rapidly evolving field to design and build biological systems solving global sustainability challenges. SGI serves three end markets: research, bioproduction, and applied products. The company's research offerings, commercialized through its subsidiary SGI-DNA, are revolutionizing science and medicine with next-generation genomic solutions, including the world's first DNA printer. SGI applies its integrated synthetic biology capabilities to reinvent bio-based production by improving existing production systems and developing novel, optimized production hosts. SGI develops its applied products, typically in partnership with leading global organizations, across a variety of industries including sustainable bio-fuels, sustainable crops, nutritional supplements, vaccines, and transplantable organs.

SOURCE J. Craig Venter Institute

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