April 15, 2016 (Vol. 36, No. 8)

Devin Leake Ph.D. Vice President of Research and Development Gen9

Multiplexing Advances Manufacturing of Synthetic Genes for Groundbreaking Research

Fueled by the race to the $1,000 personal genome, sequencing technologies have improved logarithmically in scale, processing time, and cost reduction, revolutionizing the life sciences industry and enabling entirely different approaches to many life science applications. In fewer than 15 years since the completion of the Human Genome Project, the time required to sequence a human genome has decreased from 13 years to about a day.1 As a result, the genomes of more than 180 different organisms have been sequenced, providing the genetic blueprints for bacteria, yeast, flies, worms, and mice, to name a few.

With the plethora of genomic data available, researchers now face the challenge of translating the billions of A’s, C’s, T’s, and G’s into the next generation of actionable scientific discoveries. With a further understanding of biological processes through sequence information, how can we leverage that sequence space to solve the world’s most challenging problems?

One answer is synthetic biology. It applies engineering principles to design or redesign biological systems for useful purposes, enables the utilization of the vast amount of available genetic information in such diverse areas as biomaterials, industrial enzymes, pest-resistant crops, and personalized medicines.

Despite advancements in DNA sequencing (reading), DNA synthesis (writing) has historically not kept pace. This has created a discrepancy of 3,000 times more DNA sequenced versus constructed, an issue commonly known as the “read-write gap.”

Synthesis of gene-length DNA constructs has traditionally started with the synthesis of individual oligonucleotides, followed by assembly into longer-length constructs, and finally clonal selection and sequencing. While high-quality and sequence-verified constructs can be achieved with these methods, the process is laborious, and errors introduced at the oligo synthesis and assembly stages impede throughput and scalability. This has resulted in a bottleneck that has prevented gene synthesis technology from reaching the Moore’s Law-like exponential growth curve observed in DNA sequencing over the years.

Next-Generation DNA Synthesis

A cost-effective and scalable means of producing gene constructs is key to making synthetic DNA a viable engineering tool. In 2013, a next-generation approach arose from the work of three leaders in fields of microelectronics, bioengineering, and genetics: Joseph M. Jacobson, Ph.D., head of the MIT Media Lab’s molecular machines research group; Drew Endy, Ph.D., associate professor of bioengineering at Stanford University, and George M. Church, Ph.D. professor of genetics at Harvard Medical School.

These pioneering scientists founded Gen9, a company dedicated to providing scientists with synthetic DNA for the construction of genes, pathways, genomes, and organisms. The company has successfully developed a technology called BioFab®. It enables the synthesis of thousands of full-length genes in parallel.

The BioFab platform begins with sophisticated design algorithms that parse the DNA construct of interest into oligo-length fragments, creating optimal design conditions for synthesis that take into account the complexity of the sequence space. Oligos are then chemically synthesized on semiconductor-based, high-density chips, where over 200,000 individual oligos can be synthesized per chip. Proprietary assembly technologies are next used to build the longer DNA constructs from the smaller oligos. This is done in a parallel fashion, so hundreds to thousands of synthetic DNA constructs are built at one time (Figure 1).

A unique aspect of Gen9’s technology is error correction. The oligo building blocks synthesized off-chip have an inherent error rate, which becomes increasingly detrimental to cost and turnaround time, especially for gene lengths that exceed 2,000 base pairs. Gen9 has optimized error correction strategies to ensure a precloning error rate that enables efficient, cost-effective manufacture of constructs that are as long as 10,000 base pairs in high throughput. Finally, next-generation sequencing technology is utilized to confirm the synthetic DNA constructs are clonal and sequence-perfect.

Figure 1. Gen9’s high-throughput BioFab process produces hundreds to thousands of DNA constructs simultaneously.

Multiplexing and the Read-Write Gap

While the next-generation approach of the BioFab platform enables tremendous scale to meet the growing need for synthetic DNA, continual innovation is necessary on the journey toward realizing Moore’s Law for gene synthesis. To further scale the BioFab platform, Gen9 recently applied multiplexing technology to long-length DNA construction by building 50 gene-length constructs simultaneously in a single reaction.

Multiplexing is achieved through advancements in sequence design algorithms, chemistry, and biology, and enables tremendous scale-down of reagents. The result is decreased costs. Reductions to a fraction of a penny per base pair are realized when the approach is fully implemented.

The application of the technology has the potential to scale DNA synthesis by nearly two orders of magnitude, or billions of base pairs, all within a single platform in a very small footprint (Figure 2). To put this in perspective, Gen9 estimates that current global gene synthesis demand is about 1 billion base pairs per year, a number that could theoretically be achieved in a week with the application of multiplexing technology. This will translate to reduced prices and broader access for scientists seeking to use synthetic genes across a number of different industries and applications.

Figure 2. In a recent announce­ment, Gen9 said that the latest version of its BioFab platform will improve gene synthesis by nearly two orders of magnitude and enable the manufacture of long-length clonal DNA constructs at prices as low as 3 cents per base pair.

A New Era for Gene Synthesis

These important technology advancements will enable researchers to discover entirely new ways to perform research. Metabolic pathway engineering is one of many fields set to benefit from increased DNA synthesis capacity, lower costs, and the ability to build longer-length constructs at scale. The design-build-test cycle is a proven method for optimizing pathways for expression and function, but the serial approach needed often leads to long, drawn-out projects, and just building these synthetic pathways (which can range from 5 to 50kb in length) can take researchers more than half a year to achieve.

By removing cost and design constraints, scientists can test multiple versions of the same pathway simultaneously, rather than serially to identify the optimal conditions much more quickly. The same principle can be applied to antibody engineering for therapeutics to explore variant combinations for desired properties, for example.

Multiplex technology will also be crucial in the enablement of entirely new uses of synthetic DNA, from antibiotic discovery to information storage. Antibiotic resistance is a growing concern throughout the world, and the discovery of new solutions to this problem has been stalled for years.

Some pharmaceutical companies are utilizing synthetic biology to design polyketide synthases (PKS), the enzymes involved in the synthesis of antibiotics, with the goal of making designer antibiotics. This, however, presents a technical challenge in that PKS are very large and complex. With Gen9’s multiplexing technology, this vast design space can finally be explored.

Another application that requires massive quantities of inexpensive DNA is information storage. DNA is already the oldest and most robust information storage device on the planet. Not only can DNA store genetic information, but like magnetic tape, it can be used to store all types of data. Unlike magnetic tape, DNA has a very high storage density, and therefore less space is needed. DNA storage can also survive for tens of thousands of years.

The technology to store any information in the form of DNA is not complex, and this application is only held back by capacity and cost. Gen9’s multiplexing technology removes these barriers and opens the doors to synthetic biology applications not yet imagined.

1. Lewis, Tanya. 2013. Human Genome Project Marks 10th Anniversary. Live Science.

Devin Leake, Ph.D. (dleake@gen9bio.com), is vice president of research and
development at Gen9.