Prime time for DNA

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Today, we focus once again on genome editing tools! But with a DNA twist this time.

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Prime time for DNA

Today I am covering not one, but two papers bringing DNA back into the spotlight for genome editing. But why switch to DNA? Let’s take a step back.

Since CRISPR-Cas9 was published, scientists have been working hard to improve it. A huge improvement was the arrival of prime editors: these tools install genome edits using a combo of a Cas9 nickase, a reverse transcriptase, and an RNA template. Unlike the OG Cas9, which relied on double-strand breaks and the cell’s repair machinery, prime editors offer a cleaner, more precise way to tweak DNA. But while improved, prime editors still have their limitations:

• Producing long prime editing guide RNA is time-consuming and expensive, and the yield isn’t great.

• The unprotected RNA strand can degrade easily inside the cell.

• It’s hard to make large modifications in the genome: prime editors are at their best with small edits.

• Reverse transcriptase (RT) isn’t perfect. It’s error-prone, struggles with processivity, and doesn’t have the best affinity for dNTPs (DNA’s building blocks), limiting editing efficiency

So, what’s the solution? Some researchers turned to DNA-dependent polymerases for the answer.

In the first paper, we’re introduced to DNA Polymerase Editors: not the most inspired name, but they are actually quite cool. Prime editors rely on reverse transcriptase to rewrite DNA post-cut. But these DNA polymerase editors swap out a reverse transcriptase for, you guessed it, a DNA polymerase, bringing a few improvements. The team used the φ29 polymerase, which is much better than reverse transcriptase: it has a higher affinity for dNTPs, better processivity, and a lower error rate. This adds up to editing rates as high as 50%, much higher than with prime editors. In addition, they were also able to insert a long sequence of DNA (132 bp) with a higher efficiency than prime editors (8% vs 6%).

In the second paper, the authors introduced click editors: definitely a cooler name. This system fuses a DNA-dependent polymerase with a Cas9 nickase and HUH endonuclease: this last enzyme “clicks” the DNA template into place by forming a covalent bond with single-stranded DNA. The big advantage here is the simplicity of the DNA template, dubbed clkDNA. This is nothing more than a strand of unmodified DNA: this simplicity allows the team to scale up and optimize the template faster. In this way, the authors routinely screened 96 unmodified clkDNA variants, showing that click editors can hit up to 25% editing efficiencies and install insertions of up to 40 nucleotides, rivaling prime editing tools.

I am very excited about these developments: I have always found mind-blowing how low the editing efficiencies are, and these DNA-dependent systems could be a major step toward pushing these tools from research labs into the clinic and beyond. Now, of course, these new DNA-dependent systems still share some of the issues most other genome editing tools have:

• Unwanted indels

• Incorporation of scaffold sequences into the genome.

• Limits on the length of DNA you can insert.

On the other hand, there is definitely room to grow here! For example, the polymerases used in these papers are basically “off-the-shelf”: we could improve these systems by designing polymerases specifically for inserting long sequences in the genome.

There is a lot more to unpack in the two papers, and you can read them here and here! They are behind a paywall, but you can message me, and I will share the articles.

In other news:

• DOX is in the eye of the beholder: In this study, the team investigated using DNA origami to deliver doxorubicin (DOX) in the eye. They tested different designs and found that one 24HB could achieve high drug loading and remained stable in water for 2 months, although it was not very stable in more complex environments. Unfortunately, the structures did not show a great cellular uptake: on the bright side, this could be improved by adding targeting molecules.

• Building up immunology: Cancer and infectious disease use similar mechanisms to evade immune detection. This perspective discusses how synthetic immunology can enhance immune system functions, focusing on the bottom-up approach of designing immune interventions from scratch using the nanotech toolbox. Fascinating!

• Waste or treat? Something a bit different than usual: this study investigated, using a multi-omics approach, fungal fermentation for upcycling food and agricultural waste, focusing on oncom, a traditional Javanese food. Neurospora intermedia drives the fermentation breaking down pectin and cellulose: the fungus converts waste into sustainable food. Pretty cool.

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