My name's Adrian Jervis. I'm a senior experimental officer here at the Synthetic Biology Research Center in the Manchester Institute of Biotechnology, University of Manchester. My key responsibilities are chassis and pathway engineering for the production of microbial strains to produce speciality and fine chemicals. Two of the key technologies, genome synthesis and genome editing, which allows us to produce efficient strains for chemical production. This is what we're going to talk about today. Genome synthesis and genome editing are core technologies that sit in the build stage in design build test cycle for chassis engineering. But crucially fed by information from the genome scale modeling in the design stage. Since the advent of DNA sequencing in the 1970s, we've been deciphering the genetic code. Initially, this was sequencing of single genes, but as the technology, capacity, and cost of sequencing has developed over the last 30 years, we can now sequence a whole human genome in just a few days and at a cost of just $1000. Now industrially relevant microorganisms have a typical genome size of less than half of 1% of a human genome, and over 65,000 microbial genome sequences are now available. This ability to read DNA has provided with us with books telling us how an entire organism functions. Although we have learned a vast number of words in these books, we are not yet able to really understand the story: how the highly complex gene networks produce a viable organism. Newer technology, DNA synthesis, has now given us the ability to write DNA. DNA synthesis technology has also undergone rapid development and reduction in cost. And now the first fully synthesized genomes have been completed. Simple viruses and bacteria, and is close to the completion of the first complete eukaryotic genome, that of the baker's yeast, the common microbial work horse. Like DNA sequencing, the new technological targets to be able to synthesize a whole genome for just $1,000. And it's projected that this will be possible for bacteria by 2025, and for yeast by 2035. This ability to read and write entire genomes allows researchers to begin to understand the blueprint of life, the underlying principle of "what I cannot create I do not understand". The understanding of the basic requirements for a genome, a minimal set of genes, will allow us to design purpose built cell factories. These chassis can be streamlined for the production of a wide range of chemical compounds to replace the petrochemical industry, drug discovery, development of bioremediation strategies, and many more applications. There are two main approaches to producing designer genomes. The first is to employ DNA synthesis in a bottom-up approach to build the genome from scratch. This is achieved by synthesizing small modules of the desired genome and then combining these in an iterative process until a full genome is constructed. The second approach is to use genome editing and the top down approach. This starts with a naturally occurring genome of a useful species, such as a bacteria or yeast, and editing that genome so as to delete any unrequired genes and pathways. These unessential genes may include those that have evolved to increase survival in specific environmental conditions they will not experience in the laboratory. The biggest landmark in the creation of a synthetic genome was achieved in 2010, when the full synthesis and activation of a bacterial genome gave birth to the first designed self-replicating organism, known as Synthia. To do this, the team of Craig Venter at the J Craig Venter institute in the United States chose a template bacterial species with one of the simplest genomes described, Mycoplasma mycoides. Synthesis began by chemically synthesizing short oligonucleotides of around 100 basis. These were annealed together to produce over 1,000 cassettes of DNA of approximately 1 kilobase in size. And then simultaneously introduced 10 of these cassettes into yeast, and exploited a natural process called homologous recombination. To combine the fragments into ten kilobase assemblies. This was followed by two more rounds of assembly with increasing size until the full one megabase chromosome was constructed. This chromosome is then isolated and transferred into an existing recipient cell. The synthetic genome essentially had the same architecture and sequence as the wild type strain other than 14 non-essential genes which were omitted. And the inclusion of several watermark sequences to allow them to distinguish the artificial genome from the natural genome. They also included a selection marker that would turn any cells blue that were replicating using the new chromosome. Phenotypic analysis of the new strain showed that, other than the blue color, it was indistinguishable in appearance and growth to the original wild-type strain, and Synthia still remains the only synthetic bacteria constructed to date, and will serve as a template for further chassis development. For many years, researchers have been able to edit genomes. However, until 2009, most of the technologies employed were typically only well-developed for a limited number of species. They're often also laborious and have limitations, such as the use of antibiotic resistance markers, the introduction of unwanted scar sequences. Or the ability to only make a single change in any one genome. Here we can see the typical process for gene knockouts, knock ins, and in bacteria, although similar strategies exist for many cell types. To create knockouts, a target gene, which is shown in blue, is typically interrupted with an antibiotic resistance gene, which is set in red. To knock in genes, shown in green, they're also included in the resistance cassette, and are likewise carried on to the genome. The antibiotic resistant cassette can then be subsequently removed, but it will leave a scar sequence. This kind of editing has been used for many years to great success. But new powerful genome editing technologies have now been developed that allow complete user-defined alterations, even allowing the change of a single letter of the genome. These new techniques including the CRISPR-Cas R system employ nucleases, which can be thought of as DNA scissors. Cas9 is a nuclease, which binds guide RNA. This guide RNA carries a short recognition sequence that perfectly matches the target on the genome, and directs the Cas9 nuclease to bind and cut at this site. With a cut genome, cells cannot replicate and will die. However, cells have a repair system, so they can rejoin the cut ends of the DNA, which will result in the addition to the substitution of a single base cut site, thus modifying the sequence, and any survivors will also include an edit. Donor DNA is also provided, which matches the two cut ends. It can also be incorporated during the repair process, or by cutting at two sites. The DNA in between can be deleted. It's even possible that multiple modifications can be made simultaneously, and the CRISPR-cas R system. The CRISRP-Cas9 system has revolutionized the speed and accuracy by which the genome editing can be implemented. Particularly in organisms that have been traditionally difficult to manipulate. As well as the successful booting up of Synthia, a number of other key studies have been carried out in the field of designer genomes Viruses typically have the smallest genomes of just a few kilobases and so unsurprisingly the first synthetic genomes produced were viral. The Poliovirus in 2002 and the bacteriophage in 2003. In 2006 an E.coli genome editing study was successful in the stepwise reduction of the genome by 15 percent without affecting the fitness of the strain in the laboratory. This provided an indication of how much additional genetic material some organisms need to survive outside the favorable conditions provided in the laboratory and also identified many nonessential genes. In 2009, an automated system for engineering genomes developed known as MAGE, in which a robotic platform can perform multiple rounds of multiplexed traditional genome editing massively speeding up and targeting genome engineering. In 2013 genome editing technology has also been used to recode the E.coli genome resulting in a free code which can then be used to incorporate non natural amino acids and other modifications into proteins. Eukaryotes are a much more difficult task with larger and much more complex chromosomes. But the first yeast chromosome was synthesized in 2014 in an ongoing international consortium known as SE 2.0. This is now nearing completion of the other 15 chromosomes and will soon produce an entirely synthetic eukaryotic genome for the first time. It's clear that we now have the technologies for building a synthetic genome via DNA synthesis or genome editing. But this is still a significant challenge only achievable by teams of skilled scientists with access to the correct technologies. However, these technologies will continue to develop and inevitably actually being a relatively routine process. The real rate limiting step in designing a full genome is our level of understanding of its complexity. Even the small genome Mycoplasma which is 500 genes operates in complex networks and pathways and our ability to understand this complexity is still in its infancy Despite these hurdles, genome synthesis and editing have allowed a leap in the engineering of microbial chassis, and in the coming years we should see an increase in the sophisticated design of genomes.
