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Synthego is making a big splash in the world of genome engineering. The company aims to give scientists everywhere access to the gene editing technology, CRISPR. This relatively new molecular tool holds enormous promise in accelerating basic science and advancing new therapies for critical diseases.

Synthego’s vision is to bring precision and automation to genome engineering with next-generation informatics and machine learning. By turning biology into an information science, Synthego aims to increase the rate at which experiments are performed, which in turn will dramatically accelerate scientific discovery. Synthego enables researchers to perform every step of a CRISPR experiment, from design to analysis, through its Full Stack Genome Engineering approach.

Synthego’s Full Stack Genome Engineering Philosophy 

The term full stack in software defines understanding, assembling, and controlling software code from basics to final products. A start-to-end assembly in biological products, however, is far more challenging.  Biology suffers from the reproducibility crisis because of the sheer amount of experimental variability and, complexity within and between samples.

Synthego has adopted the full stack philosophy and redefined it for CRISPR experiments by standardizing every step of the process: design, edit, and analyze. The RNA factory produces synthetic RNA guides at high throughputs. Every step of the process is monitored, controlled, and adjusted to minimize variability, improve quality, and provide large quantities of material for downstream experiments.

Synthego has also built a high-throughput and automated system for cell engineering that allows the production of edited cells to be scaled by orders of magnitude. Rigorous optimization of protocols and continuous learning feedback loops ensure that quality and predictability are on a continuous upward trajectory. Rule sets track the effect of chemistries and sequence variation on editing outcomes and are subsequently used to optimize experimental protocols. The ultimate goal is to understand and even predict the functional implications of specific editing events.

The Beginning: From Aerospace to Biotechnology

Former employees of SpaceX, brothers Paul and Michael Dabrowski, were responsible for digital design and software engineering on the rockets that would go to the International Space Station (ISS). Around this time, they saw a gap in biology and started to think about how they could apply the engineering principles that they had learned in the aerospace industry to technologies in underserved life sciences fields.

“When you look at the way scientists perform experiments, it becomes clear that a marriage between science and engineering is needed,” explains Kevin Holden, PhD, head of science at Synthego. “Most science is still performed manually—for example, pipetting—slowing the pace and making many published methods difficult to reproduce. When I worked in method development, often I would find what I thought was a great method, then not be able to get it to work in my laboratory.”

This life sciences conundrum eventually led Paul and Michael to examine how they could standardize biological workflows using automation, bioinformatics, and computational methods.

At the same time, CRISPR was emerging as an exciting, accessible technology. Due to rapid adoption by the scientific community, innovation in CRISPR progressed quickly. One such innovation was the way in which guide RNAs are produced. Rather than relying on cells to make RNA, the oligos could be made synthetically using chemical methods. But these lengthy 100-nt guides were difficult to synthesize. Materials were costly, and delivery to the customer could take up to a month, according to Dr. Holden.

Concentrating on this challenge, Synthego was born, along with a new architecture for RNA synthesis. The well-known RNA chemistry is effective; it did not need improvement. Instead, innovations in microfluidics and other processes to control the chemical reactions were needed. That led to a software platform and the RNA factory, and eventually to an early access program.

Dr. Holden reached out to key opinion leaders specializing in CRISPR who were trying to get the new guide RNAs—or were buying them at a crazy cost. One of these opinion leaders, Matthew Porteus, MD, PhD, a principal investigator at Stanford University, would later join Synthego’s advisory board.

The Birth of CRISPRevolution

Although CRISPR experts were excited, they were also skeptical about Synthego’s claims. However, Synthego met their expectations. Variables and risks were minimized by automating workflows, precisely controlling quality at each step, scaling for massive experiments, and reducing cost structures. CRISPRevolution—a portfolio of synthetic guide RNA products—helps researchers to edit cells more efficiently, including difficult cells like T cells and stem cells.

“Production from the initial RNA factory grew to generating amounts up to gram scale, including chemically modified and custom versions,” states Dr. Holden. “Since our start, we have synthesized hundreds of thousands of sgRNAs and gRNAs for a host of nucleases.”

CRISPRevolution democratizes genome engineering tools for researchers, increasing the accessibility and adoption of CRISPR techniques. By offering high-quality products with fast turnaround times, Synthego provides every scientist the ability to accurately and successfully conduct genome editing experiments.

In 2017, an investment from Intel, its first in the biotech space, helped Synthego internally develop and vet a series of bioinformatics tools to enable project and product design as well as to analyze CRISPR editing quickly and efficiently. These computational biology and bioinformatics tools are now available as open-source versions for the CRISPR community to use.

“If you want to do CRISPR in some type of automated fashion in a highly parallel way, it is important to have an efficient and reproducible synthesis process. After CRISPRevolution, we launched Engineered Cells, an automated platform for performing CRISPR-Cas9 editing in cell lines. This helps us to standardize the process,” says Dr. Holden.

Introducing Engineered Cells

Although CRISPR-based genome engineering is simpler than previous gene editing methods, the workflow associated with generating a CRISPR-mediated knockout still requires a significant amount of work. The Engineered Cells product portfolio enables scientists to bypass the setup, optimization, and execution of CRISPR experiments altogether. Through simple online ordering, Synthego offers Knockout Cell Pools and Clones in any human cell line with a guaranteed CRISPR knockout. For ultimate customization and flexibility, Synthego offers Advanced Cells. Edits include adding a gene tag, introducing a single nucleotide polymorphism (SNP) or a codon change, or editing a difficult cell line.

“Synthego’s Knockout Cell Pools take the complexity out of CRISPR with the first-ever solution that provides guaranteed results in hundreds of human cell lines. In as few as four weeks, a Knockout Cell Pool arrives with a guaranteed 50% or greater protein reduction. The knockout pools can achieve higher than 90% editing efficiency, and can be used directly in assays or to easily isolate clonal cell lines,” says Dr. Holden.

Synthego’s guarantee of a successful edit, combined with simplified access to CRISPR, eliminates the hurdle of learning new methods and optimizing protocols. The Engineered Cells products can also enable those with CRISPR expertise to expand their bandwidth quickly and efficiently.

The Process: More About Engineered Cells

Design & Generation of Guide RNA

Guide RNA sequences are designed using the Synthego Design Tool. Guide sequences are selected based on maximal homology to the primary and alternative transcripts, a high predicted on-target score, and a very low probability of off-target effects. Guides are then synthesized as chemically modified single guide RNAs (sgRNAs) through a proprietary synthetic process.

Synthego has found that guides delivered to cells in an RNA format are superior to those in a DNA format (plasmids). Additionally, guides made synthetically are more efficient than those made using in vitro transcription (IVT). The synthetic process also facilitates chemically modifying guides at specific nucleotide residues. These protect the RNA from intracellular degradation, which is a particularly important issue when working with primary cells. The combination of robust guide design and synthetic production results in the highest likelihood of generating successful knockouts.

Optimization: More than 200 Conditions Assessed

Optimal transfection conditions vary with cell type. To identify the best editing parameters, more than 200 conditions are assessed to optimize every cell line before editing. A high-throughput automated platform enables optimization in a very short time, often within a few days. This process has proven to drastically improve the knockout (KO) score even in cell lines that are traditionally considered difficult to transfect.

Cell Transfection

To generate the edit, the CRISPR-Cas9 machinery is delivered into cells as ribonucleoprotein (RNP) complexes, with synthetic sgRNA already complexed with Cas9. This format allows for maximal editing efficiency while also reducing off-target effects.

Several guides are tested at the same time, and knockout (KO) scores are determined. KO scores are a metric of knockout efficiency correlating with the proportion of sequences successfully edited in the sample pool. The higher the KO score, the more likely the guides are inducing a loss-of-function mutation. The cell population with the highest KO score is then expanded for further quality control, including a freeze-thaw viability check, mycoplasma testing, and another confirmation of the knockout-inducing indels.

One benefit of RNP delivery is high efficiency without the need for use of selection agents. While antibiotics and other selection agents are often used to enrich transfected cell populations, this also introduces selection-mediated off-target effects that can change the phenotype of cells and even negate knockouts created by CRISPR/Cas9.

KO score
The KO score (proportion of indels that cause a putative knockout) for 200 different conditions in difficult-to-edit THP-1 cells. The KO score was improved from 7% (standard protocol) to 72% after Synthego’s CRISPR knockout optimization. More than 200 conditions are tested in parallel to generate the highest possible knockout frequencies. Synthego guarantees at least a 50% reduction in protein levels, and commonly see knockout efficiencies of 75% or greater.

Analysis of Edited Cells

Once cells have undergone editing, genomic DNA is extracted and the edited region is PCR-amplified. Sanger sequencing of the resulting PCR product is then performed, and the data are analyzed by comparing to control cells using Synthego’s free and open-source Inference of CRISPR Edits (ICE) software, which facilitates fast and reliable detection of indels.

By comparing the sequencing data of control and edited cells, ICE calculates the percentage of DNA that has been edited (ICE score), the frequencies of each indel type, and the KO score. This methodology is much more precise and informative than enzymatic methods such as T7E1/Surveyor, and easier, quicker, and more affordable than next-generation sequencing.

From the ICE analysis, the guide that generated the best editing efficiency and KO score is determined, and this population of cells expanded. The final QC includes Sanger sequencing, ensuring viability is maintained post-thaw and confirming that the cells are free of mycoplasma.

Editing efficiency was optimized by testing 200 conditions editing the RELA locus in over 100 cell lines. The settings with the highest indel efficiency in addition to viable cells are chosen as the protocol to edit that cell line. Twenty-four common cell lines are depicted. Editing efficiency was calculated by PCR amplification of the cut site, and Sanger sequencing was followed by ICE analysis.

For clonal cell lines, single cells are isolated, and clones are left to expand. Once single cell clones are propagated, the genotype of each is confirmed. Two clones are delivered as standard, but more may be available by request. Each clone is sequence-verified and tested for mycoplasma and post-thaw viability.

Engineered Cells come with quality guarantees, allowing scientists to focus on generating and publishing results, rather than on developing the cells required for their experiments.

Experts Help Determine the Way Forward

Synthego’s advisory board comprises Sir Andrew Witty (former CEO of GlaxoSmithKline), Jennifer Doudna, PhD (co-inventor of CRISPR), and Matthew Porteus, MD, PhD (co-founder of CRISPR Therapeutics).

Sir Andrew Witty advises Synthego on how biopharma giants can integrate genome engineering into the drug development process. Dr. Doudna guides the standardization and innovation of CRISPR products and workflows, furthering Synthego’s mission of increased accessibility. Dr. Porteus brings his expertise in applying the genome editing technology to therapeutic development. His laboratory is working to launch Stanford’s first clinical trial using CRISPR for a potential treatment designed to treat sickle cell disease.

Supporting Innovation in the CRISPR Community

In February 2019, Synthego announced its Genome Engineer Innovation Grant program to enable leaders in the field to make big discoveries. As part of this inaugural program, up to $1,000,000 in CRISPR products will be awarded through multiple merit-based grants.

This grant enables access to Synthego’s powerful suite of genome engineering tools to help the recipients finish their current research projects more efficiently, expand their current research, or explore a new research direction.

Award amounts will vary based on the Synthego product chosen for the grant award. The deadline to apply is March 29, 2019. Winners will be announced on April 19, 2019.

The Future Roadmap

Synthego has ambitious expansion plans that include extending its Full Stack Genome Engineering platform, growing its CRISPRevolution and Engineered Cells product lines, and increasing its global footprint.

Dr. Holden summarizes: “Expect new technologies. There are a lot of interesting prospects in this space where we believe we can be disruptive and provide a great benefit or service. You will also see smarter, better ways to do CRISPR. Stay tuned.”

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