CRISPR Delivery: ENVLPE Boosts Gene Editing Efficiency 4x!

Gene editing systems are one of the hottest topics out there. One of the roadblocks? Delivery to cells. But maybe we have the solution: ENVLPE increases efficiency delivery up to 4x!

Let’s dive right in.

CRISPR Delivery Gets a Boost

It has been a while since we’ve talked about gene editing. So, today I am fixing that, bringing you ENVLPE, a smart platform for delivery of CRISPR-based gene editing systems! And deliver it does, with a 4x better performance over prior state-of-the-art methods.

The CRISPR Delivery Problem

CRISPR-Cas systems have revolutionized molecular biology. And now, these gene editing systems are moving into the clinic. This is great, but there is a big catch: delivery. An ideal delivery system would be efficient, versatile and safe, and, well, today’s systems don’t exactly excel. There a bunch of different methods, all with their own strength and weaknesses. Some of the most common are:

• Viral vectors: Maybe the most common in vivo delivery. Adeno-associated viruses, for example, are safe and effective, but they have low cargo capacity, are potentially immunogenic and they persist long, risking unwanted mutations.

• Lipid nanoparticles: Effective, just look at the COVID vaccine. The main drawback is the lack of cell-type specificity: different formulations can solve this, but the complexity increases.

• DNA nanostructures: I had to put them in. They are not as common as other methods, but they are easy to functionalize, biodegradable and biocompatible. The problem? Scale up, degradation, risk of immunogenicity (but maybe not? People are investigating).

• Virus-like particles (VLP): Virus-like particles are similar to viruses, but without genetic material, so they are not infectious. They are pretty cool, since they can target cells or tissues, and they remove the risk of mutations due to viral components. Very promising, but low stability of the cargoes and complicated design keeps their use low.

So, lots of choices, none ideal.

Introducing ENVLPE: Smarter CRISPR Delivery

To overcome these challenges, today’s paper introduces ENVLPE, short for Engineered Nucleocytosolic Vehicles for Loading of Programmable Editors. A mouthful for sure, so I’ll stick with ENVLPE.

ENVLPE (and derivatives, they’ll come up later) is a modular VLP system. It uses engineered component to actively shuttle gene-editing ribonucleoproteins (RNPs) into the nucleus of target cells. This is in contrast with other methods, which rely on diffusion to transport DNA/RNA or RNPs, reducing the editing efficiency.

How does it work? There are a few innovations (It’s a paper in Cell after all, they are always extensive):

1. Aptamer-Base Loading:

   Instead of complicated fusion of Cas9 to viral components, the system uses aptamer-tagged guide RNAs and a coat protein fused to a modified Gag protein. In this way the VLPs selectively package fully assemble Cas9 RNPs via aptamer-protein interactions, improving editing specificity and efficiency.

2. Csy4 for RNA Stabilization:

   Prime editing guide RNAs (pegRNAs) are fragile. To improve their stability and editing performance, the system uses Csy4, a bacterial RNA endonuclease, to protect the 3’ end of pegRNAs from degradation.

3. ENVLPE+ Optimization:

   Since they were not satisfied, the authors also built an enhanced version, ENVLPE+, with added domains to improve VLP assembly and the efficacy of delivery. This version outperformed earlier systems across the board!

4. Minimalistic Design:

   But what if you want a smaller system? miniENVLPE is there for you: the team also created a stripped-down version. This variant removes unnecessary viral elements, reducing complexity while maintaining efficacy.

Applications and Results

I know what you are thinking: this is cool, but did it work? Well, to answer that the researchers extensively validated the system, in vitro and in vivo.

1. Testing Different Editing Modalities:

   • Prime Editing (PE): Just as a reminder (for myself), prime editing requires just a single nick in the DNA, instead of a double stranded break of traditional CRISPR systems. A reverse transcriptase then creates a DNA strand from the pegRNA. They tested a both PE2 and PE3 systems.

   • Base Editing (BE): The team tested cytosine and adenine base editors.

   • Cas9 Nuclease Editing: Good old system for targeted knockout and homology-directed repair (HDR).

2. Results in Different Systems:

   The team tested the performance in quite a few systems!

   • HEK293T cells and iPSCs: ENVLPE+ achieved superior editing efficiency, when compared to previous state-of-the-art systems, up to 4x more!

   • T cells: They achieved a robust editing in human primary T lymphocytes, without toxicity. Also, I discovered that T cells really don’t like to be modified with dsDNA, but they like RNPs or ssDNA more.

   • Mouse Retina Models: ENVLPE also works in vivo. The team showed functional gene restoration in mouse models of inherited retinal disease after subretinal injection. Pretty cool!

   • HDR Enhancement via IDLV: Combining ENVLPE with lentiviruses carrying DNA templates, the researchers showed successful homology-directed repair in reporter cell lines.

To Conclude: Strengths, Limitations and Future Work

Okay, time to finish: ENVLP and variants represent a big step forward for the delivery of gene editing systems. They bring a lot of strengths:

• High Editing Precision: The systems showed minimal off-target effects despite high on-target activity.

• Payload Versatility: Works with nucleases, base editors, and prime editors.

• Programmability: Easily customizable by changing guide RNAs or surface proteins (maybe using custom nanocages?).

They are still not perfect though:

• Scalability: Like always, large-scale VLP production and purification remain a hurdle.

• Editor Stability: Improvements may be needed to stabilize complex editors like prime editors for certain cell types.

• Targeting: There is a degree of targeting flexibility, but receptor expression and efficiency in diverse tissues need further optimization.

This was a great paper! And if it’s not clear by now, the work is extensive: I honestly could not fit everything here, so go and read it yourself here!

If you made it this far, thank you! What do you think of ENVLPE? Did I miss something important? Do you see some more potential or problem with this system? Reply and let me know!

More Room:

• Making Origami with mRNA: Feeling like combining DNA and mRNA origami? You are not alone. This study presents a modular gene delivery platform using mRNA–DNA origami, where mRNA is folded into structured nanocarriers with DNA strands. The researchers show that preserving ribosome-binding sites is key to maintaining translation efficiency. Encapsulating these structures in virus-like capsids protects them from degradation and improves cellular uptake. The work offers valuable design insights for developing more stable and functional mRNA-based therapeutics.

• Nanotags and Cryo Microscopy: DNA nanostructures are very good at precisely positioning ligands. This article highlights how this characteristic of DNA nanostructures can be used to create nanotags for electron microscopy, particularly cryo-ET. They could enable multiplexed analysis and in situ particle identification, but challenges remain in delivering them into live cells. Promising strategies include direct cytosolic delivery and RNA-based expression, though more research is needed to fully understand their intracellular behavior.

• DNA NanoFireworks for Cancer Therapy: Not many names better than nanofirework! This study presents a self-assembled DNA nanofirework for amplified imaging of microRNA in living cells. The nanostructure enables efficient cellular uptake via endocytosis and offers strong stability and biocompatibility. It combines DNA nanotechnology with catalytic amplification to detect miRNAs at low concentrations. This platform offers a powerful tool for live-cell RNA imaging, advancing research into disease mechanisms and potential RNA-based therapies. And an amazing name!

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