Evolving aptamers with CRISPR-Cas

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Today we are back talking about genome editing, with a cool CRISPR-Cas based system!

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Evolving aptamers with CRISPR-Cas

I have actually not written enough about gene and genome editing, so it’s time to fix that! And what better way to do that than to jump straight into a CRISPR-Cas system?

But let’s start with the basics. Genome editors work by creating double-stranded breaks (DSBs) at precise locations in DNA, using programmable nucleases. After the break, the cell’s natural repair mechanisms step in, allowing researchers to introduce targeted modifications, such as insertions, deletions, or even precise rewrites. The system is made up of 2 key components:

• Cas nuclease: a CRISPR-associated proteins (or just Cas), like Cas9, which cuts DNA at a specific site.

• Guide RNA (gRNA): a customizable RNA sequence that directs the Cas protein to a specific DNA target.

This minimalistic yet powerful setup is why CRISPR-Cas dominates genome editing, being precise, adaptable, and quite easy to modify. For example, often researchers use a non catalytic version of Cas9, called dCas9, when they just want the protein to bind to the DNA without cutting. By attaching additional tools to dCas9, like base editors (to tweak individual bases) or prime editors (to rewrite DNA), researchers can achieve incredibly specific edits!
At the same time, it’s also possible to modify the gRNA to add RNA aptamers, short RNA sequences capable of binding to protein. These modified gRNAs allow us to recruit RNA-binding proteins (RBPs) to precise DNA locations, enabling targeted gene regulation. This opens up possibilities for multiplexed editing, where different aptamers (designed to work orthogonally) simultaneously regulate multiple genes without cross-talk. Unfortunately, current RNA aptamers systems have some limitations:

• Lack of Orthogonality: Only a few aptamer-protein pairs exist, and most are prone to unwanted interactions.

• Inefficient Discovery: The dominant aptamer discovery method, SELEX, is time-consuming and often fails to produce aptamers that function well inside cells.

This brings us to today’s paper. The researchers saw all of this, and created CRISPR-Hybrid, a platform designed to evolve aptamers directly inside of cell. This ensures that the aptamers are functional in the environment where they’ll be used. Very cool idea! And equally cool name. The system combines programmable DNA targeting with RNA-protein interactions for precise and selectable transcriptional control.

This CRISPR-Hybrid platform has a few key components:

• dCas9: A catalytically dead Cas9, which binds to the DNA without cutting it, and it allows to target the recruitment of molecular complexes.

• Single guide RNA (sgRNA): This is the targeting system, but it’s also extended with a randomized aptamer library, providing the base materials to be evolved.

• Reporter gene system: A fluorescent reporter protein (of course it’s GFP) downstream of the dCas9 binding site acts as a readout for the functionality of the aptamer: activation of the fluorescence means that the aptamers works to recruit RNA-binding proteins.

• Target RNA-binding protein (RBP): An exogenous RNA binding protein is fused to a transcriptional activator: in this way, when the aptamer binds to the RBP the reporter gene can be activated.

• Fluorescence-based selection: Last but not least, cells expressing functional aptamers are sorted using fluorescence-activated cell sorting (FACS).

Once these components are in place, the mechanism is simple:

1. Initialization: The sgRNA library with randomized aptamer sequences is expressed in cells alongside dCas9. The dCas9 binds a target DNA site near the reporter gene, positioning the aptamers for evaluation.

2. Activation and Selection: If an aptamer successfully binds the RBP-transcriptional activator, it drives GFP expression. Brightly fluorescing cells indicate functional aptamers.

3. Iterative Evolution: High-fluorescence cells are isolated via FACS, and their aptamer sequences are sequenced. This process is repeated to enrich high-affinity aptamers!

So this was the idea! And what were the results, you ask? Well, the researchers used this system to screen a randomized RNA library to evolve aptamers for the Qβ coat protein (QCP). And they identified A9, an aptamer with high affinity for this protein and low cross reactivity with other RBPs! To validate this new aptamer, they tested this A9-QCP aptamer-protein pair in both bacterial and mammalian cells, showing a robust functionality. And, more interesting, they showed that this pair can be used together with a different pair to achieve multiplexed gene regulation, where one gene is activated using the A9-QCP pair while another gene is repressed using a different pair! Super cool.

This was a very cool paper, from a field I am still learning about. I had to skip on a lot of interesting discussions and optimizations the researchers had to do, so I strongly recommend you go and read the paper here! This or similar technologies hold great promises for therapeutics and synthetic biology.

And as always, thank you for reading!

More room, more news:

• More than peas to Gregor Mendel: We have all studied how the work of Gregor Mendel started modern genetics, and most of us don’t remember much more than his peas. But this article in Asimov Press reveals a lot more nuance in the life of the friar: for example, after the Mendel’s death over a 1000 pages of his work was burnt, for whatever reason. And there is a lot more, so go and read it! It’s very cool.

• The nitroplast: the latest addition to organelles: We all remember how the mitochondria and the chloroplast evolved from the integration of bacteria into eukaryotic cells. Now, another members is added to this exclusive club: the nitroplast. Studying a marine alga, researchers in this paper reported the discovery of a new organelle which is capable of nitrogen fixation, fundamental for algae and plants. Time for me to back to study basic cell biology!

• More DNA crystals: If you like DNA crystals, this is the review for you. It highlights recent progress in 3D DNA crystals, macroscopic materials created through DNA self-assembly. The review categorizes crystal structures by bond orientations, examines in-silico design and self-assembly, and explores applications in biosensing, biocatalysis, DNA computing, data storage, and more. Very useful!

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