After over three years of super stealth mode, Inscripta gave its first public presentation today at the 2019 Synthetic Biology: Engineering, Evolution & Design (SEED) conference in New York City. Inscripta offered a peek into their progress toward making “the world’s first scalable platform for benchtop digital genome engineering.”

“We want to unleash researchers’ imagination so that research is not limited by the tools,” notes Kevin Ness, PhD, CEO of Inscripta. To do that, they are addressing the challenges and limitations that exist in the CRISPR space today. “The tools are inadequate to realize the full potential of what CRISPR can do,” Ness tells GEN.

From well to cell

Currently, Ness explains, a scientist has to prioritize in the experimental design of their genome editing projects, which is limiting. They can choose the number of locations in the genome to edit, or the complexity in the type of mutations. Ness notes that researchers can have “a flavor of richness” in the type of mutations made at one site in the genome. Or, they can make one type of change in many places—like George Church’s, PhD, professor at Harvard Medical School, recent milestone of making simultaneous edits to more than 10,000 loci in human cells. Combining these two cannot be done. The CRISPR world is stuck in one of these two lanes.

Inscripta’s goal is to facilitate making multiple types of mutations in multiple sites in the genome. To do this, they have moved the partitions that separate experiments away from the well of a 96 well plate—to the cell.

The technology that is at Inscripta’s core was first published in a 2016 Nature Biotechnology paper titled “Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering” from the lab of Ryan Gill, PhD, associate professor of chemical and biological engineering at the University of Colorado, Boulder. The first author on the paper, Andrew Garst, PhD, co-founder and principal scientist at Inscripta, and the team from the Gill lab write that “CRISPR-Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale.” They call the method “CRISPR-enabled trackable genome engineering” or CREATE. The key is the covalent attachment of each guide RNA to homologous repair cassettes. Inscripta builds libraries of these cassettes on a microarray, each of which has a barcode so that each cell can be tracked. How does Inscripta ensure that only one CREATE cassette—one molecule—gets into each cell? That is, according to Inscripta, proprietary information that involves a microfluidic system in the machine. Or, as Ness describes it, part of Inscripta’s “special sauce.”

Lysine biosynthesis at the cutting edge?

Presenting what Ness calls a “tease of the type of data that their platform can generate” was the job of Richard Fox, PhD, executive director of data science at Inscripta. Originally trained as a nuclear engineer, Fox started with a story of where he was when he found a copy of “The Selfish Gene” by Richard Dawkins—on an aircraft carrier in the Pacific. The book not only changed his career path but changed the way he thought about the world. After some time, he found himself “playing around” with proteins using directed evolution. Indeed, Fox is the inventor of the protein sequence activity relationship (ProSAR) algorithm, a directed evolution approach with model building and library design, that can be applied to protein engineering.

Like his work in the protein field, Fox’s role at Inscripta focuses on improving biology through engineering—specifically the challenges of “genome scale engineering.” In proteins, Fox notes, this process is well developed. However, he was confronted by a new challenge in asking the question, “How do we engineer pathways and genomes to harness the power of biology?” He knew that part of the answer was something he was familiar with—diversity generation and large scale, multiplex combinatorial editing. He also knew that what they wanted to do required a greater scale and broader variety of edit types than was currently possible.

Fox and the team at Inscripta turned toward evolution as their inspiration. They favor the idea of generating diversity and then recruiting the benefits into combinatorial optimization over taking iterative steps. To go after diversity, they needed to go after the entire genome—large edits at genome scale. Using these foundations, Inscripta harnessed the Lysine biosynthesis pathway. A classic metabolic pathway, they asked how they could make directed mutations to improve the phenotype of Lysine production. To do this, they generated diversity in the Lysine pathway on a genome-wide scale. With a total of 200,000 edits, which Fox calls “transformational editing capability,” they hit every gene in the genome, with “non-trivial” mutations. These, he defines as insertions, deletions, and swaps including variable promoter ladders.

They then went on to measure the amount of Lysine produced by the mutants using a Rapid Fire Mass Spectrometry, with reads every seven seconds. He notes that since CREATE “unleashes a torrent of edited cells” a quick phenotypic readout is critical.

As expected, mutations in dapA, a key enzyme in Lysine biosynthesis were identified. They also identified other known genes in the pathway and new targets outside of the pathway. A number of those genes are uncharacterized and some even remain unannotated in the E. coli genome.

Finishing with a glimpse into Inscripta’s future, Fox showed how stacking the identified edits improves lysine production. When they built a library on top of the engineered strain, they saw fold increases of Lysine production by some strains. And, in combining just two edits, a strain had 10,000-fold increase of Lysine production over the wild type, illustrating the power of their approach.

There are a lot of details still under wraps, despite the launch of their platform set for later this year. And, big claims. “Inscripta will do for genome writing what Illumina did for genome reading,” asserts Ness. (To be fair, he did start 10X genomics “out of his garage.”) He tells GEN that their more than 100 employees—“the best development team that has ever assembled in the life science space”—are happy to be “tool providers.” They are not interested in asking the big scientific questions themselves. Rather, their goal is to enable scientists to move beyond where CRISPR is now and drive science in profound ways. Because, he asserts, “As exciting as CRISPR is right now, it’s not enough to get us there.”