While scientists can read DNA fast, editing and writing it is not that easy. Increasing the speed of editing and writing DNA sequences will be critical to ushering in new applications of genome engineering.
Much of biotech is about reverse-engineering the designs that billions of years of evolution have put together. To be able to do so, companies need tools that allow us to read and write the language of life. DNA sequencing technologies, with a few decades of developments behind them, are becoming cheaper and faster than ever before, allowing us to read massive amounts of genetic information. Techniques to write and edit this language, on the contrary, are relatively newer, more expensive, and slower.
DNA editing and synthesis technologies allow biotechs to make precise edits in the genome and synthesize genes from scratch, respectively. These technologies are the backbone of a new synthetic biology paradigm that seeks to advance genetic engineering beyond the copy-and-paste paradigm of early GMOs. However, the slow speed of DNA editing and writing technologies, more than anything else, poses a bottleneck to this transition.
Multiplexing with CRISPR gene editing
For researchers studying gene function, gene editing technologies provide a way to target specific locations in the genome. However, most diseases are determined by multiple genes.
Over the last decade, researchers have put CRISPR-Cas9 gene editing to use in a range of genome engineering applications. The gene-editing tool can be directed to specific locations in the genome by using guide RNA molecules that match the target sequence, instructing the Cas9 enzyme to cut the DNA. This enables multiplexed gene editing, which consists of editing multiple locations at once by providing several different guide RNA molecules simultaneously.
Swiss biotech Cytosurge, for instance, leverages multiplexed gene editing to generate cell lines for gene and cell therapies as well as bioprocessing. Cytosurge CSO Tobias Beyer described the company’s technology as “a combination of atomic force microscopy and fluidics. The precision of this technology makes it possible to deliver the same amount of Cas9 protein and guide RNA per cell. Because you can control the amount of Cas9 going into the nucleus, you can use as little as needed to make the cuts.”
Reducing the amount of guide RNA and Cas9 protein entering the cell can reduce the likelihood of off-target edits. According to Beyer, when you want to produce a protein using a culture of gene-edited cells, it doesn’t matter what off-target effects take place as long as it gives you the protein you want with the highest purity. However, when studying how specific gene edits affect a cell’s behaviour, these off-target effects can severely distort results.
Modulating expression of multiple genes
Gene editing goes beyond knocking out or introducing one or more genes. Modulating the activation of genes allows researchers to study a lot more interactions between genes and their transcriptional factors.
“In addition to multiplexed genomic changes, we have developed CRISPR-based technologies that activate or interfere with gene expression. These do not cut DNA and are ideal for multiplexing,” said Emily Anderson, Principal Scientist at Horizon Discovery, a UK-based company that provides gene editing and gene modulation services.
“The ability to edit or modulate multiple genes simultaneously offers tremendous advantages to researchers who want to model and develop therapies for complex disease landscapes beyond monogenetic disorders like cystic fibrosis or sickle-cell anemia.”
Multiplexed gene editing can also be applied to the development of gene and cell therapies, including CAR-T cell immunotherapies. Multiplexing also speeds up the development of gene-edited cell lines as compared to making edits one at a time. “For immortalized cells, the process of clonal selection and characterization is arduous, resulting in lengthy timescales. Going through it once is hard enough, and going through it for multiple rounds compounds this issue,” Anderson told me.
Synthesizing long DNA sequences
Synthetic biology is the basis for many biotech applications. Some examples are medical research or using DNA as data storage. But a big limitation in this research field is that conventional DNA synthesis technologies are unable to write DNA sequences over a few hundred nucleotides long.
“The conventional way of DNA synthesis as a sequence of chemical reactions to attach one base to the next step-by-step has a certain error rate and limits the length of DNA molecules that you can make. The other way is using enzymatic approaches, but they are also limited in producing long DNA strands,” said Marc Brehme, CTO of Ribbon Biolabs. This Austrian biotech startup employs multiplexed parallel assembly to create long strands of synthetic DNA.
“The error rate in conventional chemical methods of adding bases adds up and accumulates the longer you make the molecule,” Brehme told me. Ribbon Biolabs uses conventional synthesis to make multiple short strands simultaneously to then stitch them together and form long DNA chains with a minimal error rate.
“At every step, we’re doubling the length of the molecule. We do that in a very controlled, compartmental and highly parallelized manner, using automated robotic automation.”
Ribbon Biolabs has already synthesized the full genome of small bacteriophage viruses. “Currently, the longest that we’ve made is 20 kilobases. We’re really focusing on scaling this technology after our Series A investment. We are now establishing a production facility, we’re expanding into the US, and making the technology ready for the market,” Brehme said.
Combining multiplexed gene editing and gene synthesis
A major application of multiplexing is the creation of large DNA libraries for running experiments with thousands of sequences simultaneously. This provides high-throughput data into how the genotype correlates with the phenotype. “When we make a DNA molecule, making a variant would require only changing a small part in the process. We can generate a lot of variants in parallel in a multiplexed manner,” Brehme told me.
On the other hand, lessons from gene editing can be put to use in error correction for DNA synthesis. For instance, base editors — a genome engineering tool that makes single-base mutations — can be used to fix errors made when creating long strands of synthetic DNA.
Lastly, the combination of multiplexed gene editing and long DNA synthesis opens up exciting avenues in biotech. Think of the rapid and on-demand development of personalized therapeutics, for example. In addition, being able to engineer large eukaryotic genomes, including mammalian genomes, will be critical to progress genome-scale projects such as de-extinction, where a species can be cloned using its genome.