We all know by now that the CRISPR-Cas system is nothing short of a revolution for gene editing, but DNA synthesis can’t keep up with CRISPR. Since these molecular scissors burst onto the scene, precisely chopping DNA wherever we choose, it’s been poked, prodded, and engineered to enable an array of editing functions; including regulation of gene expression, base editing, and even whole gene deletion and insertion.
CRISPR-based technologies stand on the brink of conquering some of humanity's greatest challenges, from curing genetic diseases and combating cancers to engineering crops that can withstand our dramatically changing climate. Yet, the full realisation of this revolutionary potential is hampered by the struggle to produce long, accurate, synthetic DNA at scale.
gSynth® is turning this on its head.
Multiplexed CRISPR-Cas editing
Guide RNA (gRNA) is the sat nav of the CRISPR-Cas system directing a Cas nuclease to make precise cuts in a DNA sequence. By supplying the Cas protein with a customised, synthetic gRNA, we can accurately cleave specific sites within an organism’s genome.
Targeting a single site with CRISPR-Cas is relatively straightforward; it requires only one, short gRNA, which is easy to synthesise on a small scale. However, multiplexed CRISPR-Cas technology takes it a step further by enabling us to target multiple genes simultaneously. This approach uses gRNA arrays, which are collections of multiple guide RNAs assembled on the same sequence. The popularity of multiplexed CRISPR-Cas editing is rapidly growing because it allows for:
- Higher efficiency: When multiple gRNAs target a single gene, the efficiency of editing or regulating that gene increases significantly. For instance, a single gene targeted by multiple gRNAs using Cas12a can reach around 60% editing efficiency compared to much lower rates with individual gRNAs.
- Multiple edits: CRISPR-Cas multiplexing enables the simultaneous editing of multiple genes, which is particularly beneficial for studying complex traits and treating polygenic diseases.
- Controlled regulation: gRNA arrays can be used for enhancing or repressing the activity of multiple genes at the same time. Similar to making multiple edits, the ability to modulate multiple genes creates new potential for studying and treating complex traits and polygenic diseases.
Many important agricultural traits and human genetic diseases are influenced by multiple genes. Multiplexed CRISPR-Cas opens up the opportunity to simultaneously edit or regulate these genes, providing a more comprehensive approach to understanding and potentially curing complex genetic conditions or improving crop traits in a single step.
There’s just one problem. That problem is DNA.
Assembling long and complex gRNA arrays is challenging since as the length of the sequence increases, the yield of error-free sequences decreases significantly. Even with the best nucleotide coupling efficiencies, when phosphoramidite synthesis reaches 200 nucleotides, only ~40% of the material is the correct full-length product. Moreover, the presence of multiple repeats can result in synthesis delays or failure and therefore, are often rejected by vendors.
Multiplexing CRISPR screens
CRISPR screens are high-throughput, genome-wide, loss-of-function studies that help us understand how changes in genes affect traits or disease outcomes as well as identify possible targets for drug discovery.
A typical CRISPR screen goes something like this:
- A sgRNA directs a Cas endonuclease to the gene of interest
- The Cas nuclease creates a double-strand break at the target site
- Non-homologous end joining cellular DNA repair pathway is activated
- If the DNA repair results in a frameshift mutation, this can knock out the target gene
- The resulting change, or phenotype, can be observed to infer the loss-of-function
Similar to multiplexed CRISPR-Cas editing, multiplexed CRISPR screens using multiple guide RNAs offer several advantages:
- A single gRNA may not cause a frameshift mutation and consequently, the target protein could retain its function. By using multiple guide RNAs to concurrently cut a gene at various locations, the likelihood of a successful knockout is significantly increased.
- Multiple gRNAs can be used to target multiple genes in a single screen identifying interactions between genes and their combined influence on certain traits or conditions.
There’s just one problem. That problem is DNA.
A significant challenge is a labour-intensive process required to synthesise and incorporate many guide RNAs into a single genetic vector using traditional methods, reducing the number of genes that can be targeted in a single screen.
CRISPR-Cas and gene replacement
Most CRISPR-Cas technology has been confined to the editing of a few bases. But, if a DNA template is also delivered, cells can incorporate a corrected copy into their genomes once the Cas nuclease has opened up the DNA.
Indeed, CRISPR-Cas tools that could potentially ‘cut’ out faulty genes and ‘paste’ in full-length copies of corrected genes have been developed. This could be used to treat genetic conditions with a large number of mutations or insert genes, like disease resistance, into important agricultural crop species.
While researchers are trying to iron out the complexities of introducing large DNA inserts into a genome, such as unintended chromosomal deletions and re-arrangements that are harmful to cells, there is one significant challenge that they all face – getting their hands on accurate, full-length gene sequences.
There’s just one problem. That problem is DNA.
With CRISPR technologies like PASTE promising to insert large chunks of DNA – upwards of 36,000 base pairs – access to accurate, gene-length DNA beyond what is possible with legacy technologies is crucial.
DNA synthesis is stifling CRISPR success
Unlike Cas nucleases, short, error-prone sequences produced by chemical synthesis just won’t cut it when it comes to advancing CRISPR-based technologies.
Even with the best nucleotide coupling efficiencies, when phosphoramidite synthesis reaches 1000 nucleotides, the error-free yield is <0.01%.
The impact? Getting your hands on gene-length sequences or long guide RNA arrays requires a lengthy and laborious process involving chemical synthesis of short oligonucleotides and assembly followed by significant post-synthesis steps to correct errors and identify error-free sequences.
Using chemical synthesis to produce repetitive sequences - like those found in CRISPR guide RNAs - results in errors, purification challenges, and often synthesis failure.
The impact? DNA synthesis providers often reject complex sequences or take months to deliver them due to technological limitations and extensive error screening. Clearly, this doesn’t scale for high-volume gene consumers such as those carrying out automated, high-throughput CRISPR-Cas screening.
Chemical synthesis requires large volumes of hazardous reagents and solvents, as well as energy-intensive processes in synthesis, purification, and isolation.
The impact? The pharmaceutical industry has acknowledged the urgency for more sustainable production of oligonucleotides and is committed to achieving net zero emissions. The reliance on chemical synthesis presents significant challenges in meeting this goal.
There’s just one solution. That solution is gSynth.
gSynth: A better way to make DNA
The inability of chemical synthesis to produce long, accurate, complex DNA means getting your hands on gene-length sequences or complex gRNA arrays is often the rate-limiting step of propelling your CRISPR-Cas research forward. Until now.
Any sequence
To overcome the limitations of chemical synthesis, DNA providers can demand you codon optimise your sequence to facilitate successful synthesis – or your sequence will simply get rejected. However, this approach does not work in all cases. For instance, genes encoding functional noncoding RNA elements, as even minor sequence changes could alter the RNA's structure and impair its function.
We’ve removed the need for codon optimisation to make synthesis work – so the only optimisation you need to do, is the optimisation that improves your research, application, or therapeutic.
Any length
As the length of a DNA sequence increases, the yield of error-free DNA decreases significantly. This means that phosphoramidite synthesis is generally limited to <200 bases.
Any complexity
DNA providers often reject complex sequences or those containing multiple repeats. If they accept, you’ll likely suffer long delays with increasingly complex sequences.
We’ve done away with the limits on complexity. No matter your length, GC content, homology, repeats, or overall complexity score, gSynth has your synthesis needs covered.
We’re on a mission to change the way you can synthesise DNA.
