Gene Editing Advancement: 200x Improvement in DNA Insertion

Gene editing had an amazing streak lately, with the first therapies in real-world patients. But it’s just the beginning, and the DNA sequences that can be inserted are very short.

Today’s paper introduce an amazing system to insert long DNA strands into the genome of human cells, using evolved CRISPR-associated transposons! And the results are amazing: 200x improvement in insertion rate over the wild type protein!

Let’s dive right in.

Evolving Better Gene Editors

Gene Editing: Promises and Limitations

Gene editing is finally stepping out of the lab and into the clinic.

Technologies like programmable nucleases, base editor and prime editors can disrupt, install or correct practically any sequence. With over 60 clinical trials underway, we are getting close to one-time treatments for genetic disorders!

But challenges remain.

These tools are great with smaller sequences (<200 bp), but struggle with inserting gene-sized DNA sequences (> 1kb). But why is it even important? Because many diseases are caused by different mutations in the same gene. And these mutations are not the same in all patients. Editing each mutation individually would require creating (and approving) many custom therapies. Expensive, slow and inefficient.

A better approach would be to insert the healthy gene, replacing the faulty version without worrying about the mutations. And this would make it a one-and-done treatment!

Why Viral Vectors Are Not Enough

Traditionally, gene delivery has relied on viral vectors. But, as we have seen before, they have limitations and, honestly, some risks:

• The DNA insertion is random and potentially oncogenic

• There is potentially a need for redosing

• Viral vectors can elicit an immune response

• Since the integration is not targeted, there can be problems with protein over- or underproduction

The ideal system would install a full gene at exactly the right place in the genome. This would cure the disease permanently, safely, and efficiently!

CRISPR-Associated Transposases (CASTs): A Different Path

That’s where CRISPR-associated transposases (CASTs) come in.

These recently discovered systems use a RNA-guided CRISPR-Cas to find a specific DNA sequence. But instead of cutting like a nuclease, they use a transposase protein to directly move DNA from a donor inside the genome. Very cool! And no double stranded breaks.

And they work amazingly! In E.coli: they really don’t work in human cells, with a pitiful 0.1% insertion efficiency.

These proteins probably evolved this low efficiency to avoid killing the host with too much DNA hopping around. But the author’s of today’s paper saw potential, and asked: can we unlock the hidden potential of these proteins?

Oh yes, we can. And they did it!

PACE: Evolution on Fast-Forward

To improve the CAST system, they used one of the coolest techniques out there: phage-assisted continuous evolution, or PACE.

PACE is a powerful method for rapidly evolving proteins in the lab. It uses a specially engineered bacteriophage (virus that infects bacteria) system where the survival of the phage depends on the activity of the protein you’re trying to evolve. If a protein variant performs better, the phage will replicate! If it doesn’t, well…

This process runs in a controlled setup, where phages constantly infect fresh bacteria, mutate, and are selected for improved functions. In this way researchers perform thousands of evolutionary generations in days, far faster than traditional methods! And the technique has few requirements: the protein just has to be expressed in E. coli. No need for structural or biochemical characterization. The sequence is enough.

The team used this system to evolve a CASTs system called PseCAST, linking transposition activity to phage propagation. And by the way, the protein system is big, like 7 proteins working together: even more impressive they made it work!

The process was not simple: the team fought with low starting efficiency, cheating phage variants, and slow integration. But they managed, and obtained different variants that they evaluated them on human cells. The team tested the insertion of a 1-kb long DNA strand: and the result was a 100x improved efficiency compared to the wild-type! But the researchers were not satisficed.

evoCAST: A CAST Built for Human Cells

The team combined the optimized CAST with a rationally improved DNA-targeting complex (QCascade) to create evoCAST, a system fully optimized for human cells.

evoCAST showed an improved of over 500 times over the wild type system, and it had no problems integrating DNA up to 15 kb! When tested on human cells, evoCAST achieved 10-30% targeted gene insertion efficiency across 14 genomic loci, including genes that are relevant for therapies, such as ALB, important for hemophilia B, or TRAC, used for immune cell reprogramming.

The team also tested the integration of DNA to directly replace diseased genes, independent of mutations. These included genes for Fanconi anemia, X-linked immunodeficiency, Rett syndrome and phenylketonuria. Lastly, evoCAST worked in multiple human cells lines, with integration varying from 1.6% to 19%.

It worked particularly well in primary human fibroblast, with up to 30% efficiency!

Conclusions and Future Directions

Such an amazing work! To summarize, the authors developed a new system to evolve CASTs towards improved activity in human cells, with up to 500 times improvement on the wild type!

It’s a very powerful system, with lots of advantages:

• Easy to reprogram

• High product purity

• Safer, since it avoids genomic double strand breaks

The authors highlight off-target insertions as a limitation, like with many other nucleases. But they can be limited using transient delivery method, such as mRNA delivery. Once the wanted integration is successful, the proteins are degraded, to avoid problems.

Gene editing can revolutionize medicine, potentially solving genetic diseases with a single treatment. And this programmable insertion could even be useful for other applications, such as CAR-T cell therapy or generation of cell lines and transgenic model organisms!

This was a great work! I had to skip lots of data. If you are curious about the technicalities of PACE, the evolution of the proteins or comparison with other methods, just read the paper here!

If you made it this far, thank you! What do you think about gene editing? Do you have a favorite system? Reply and let me know!

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• DNA Origami Gets the Golden Treatment: You know what else I find cool, aside from nanomedicine? Nanofabrication! This study expands DNA origami placement to gold substrates, overcoming previous limitations. Using microcontact printing of self-assembled monolayers (SAMs), the researchers created patterned Au surfaces with distinct surface chemistries to guide selective DNA origami binding. Monte Carlo simulations informed optimal conditions, and the use of bifunctional SAMs enabled high-yield, site-specific placement. This paves the way for integrating DNA nanostructures into a wide range of device platforms.

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