Can DNA challenge RNA’s dominion over CRISPR-Cas, and secure a place in the genome editing world?
Today’s paper turns Cas12 from a DNA-cutting enzyme into an RNA-sensing platform. All thanks to DNA’s power! So yeah, it’s DNA’s time to shine.
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DNA Comes for CRISPR
Researchers created a new type of guide DNA for Cas12, expanding the enzyme’s editing potential to RNA.
CRISPR-Cas 101
Genome editing is changing biology.
Well, it already did it. It’s everywhere in biological research; there are a thousand different versions; and the first personalized therapy saved a baby’s life last year! The most famous one?
CRISPR-Cas, of course.
At its most basic, CRISPR-Cas systems have two main parts:
Cas effectors: Enzymes that bind and cut the target → nucleases.
crRNA: The RNA that recognizes a specific sequence on the target. Generally, it’s incorporated in a guide RNA (gRNA).
This is the base, but there are many flavours! Some are natural, and some are engineered. A few examples:
Cas9 and Cas12 → the classics; they target DNA and create double-stranded breaks.
Cas13 → targets and degrades RNA.
dCas9 → a deactivated Cas9 that still binds DNA, making it useful as a blocker or as a targeting module for fusion proteins, like epigenetic editors.
And these proteins have a quirk that makes them perfect for nucleic acid diagnostics: collateral cleavage.
Here’s how it works:
The Cas-crRNA binds its target DNA/RNA.
The enzyme is activated.
It starts cutting nearby reporter DNA/RNA molecules.
These reporter molecules carry fluorescent or colorimetric labels, so when Cas cuts them, you get a visual signal. A smart trick: you use non-specific activity to create highly specific detection in little time!
crRNA: Modular DNA Targeting
But enough with Cas proteins!
The crRNA gets a lot of attention too, and rightly so. It’s the other half of the system, after all! And it’s not just a simple targeting tag. In many systems, crRNAs have a scaffold region with stem-loop architecture that is essential for binding DNA/RNA targets and stabilizing the complex.
Because of this, scientists have engineered many improvements:
modified bases
Chimeric DNA+RNA versions
Longer, shorter, and everything in between.
Why bother? Well, to improve gene editing efficiency, stability, and shelf life.
But it’s still RNA! It struggles with:
Complexity → RNA is not super easy to synthesize
Cost → Complex things are expensive
Stability → RNA’s shelf life is shorter than other biological molecules
You know what’s similar to RNA, but doesn’t have these problems?
DNA.
But Marco, DNA doesn’t work with Cas! You need RNA! Well, about that…
ΨDNA: Pseudo-Guide DNA
Let me introduce ΨDNA, the protagonist of today’s paper.
ΨDNA (pseudo-guide DNA) is a DNA-based guide that lets Cas12 nucleases target RNA instead of DNA. The authors reversed the usual crRNA logic, building a DNA mimic of the crRNA scaffold!
And it works. Cas12 can use ΨDNA to target RNA and still trigger collateral ssDNA cleavage. Exactly what you want for detection!
But do you think they stopped there? Oh no.
ΨDNA also works in cells:
It repressed translation and knocked down cellular RNA
It supports DNA editing and RNA knockdown at the same time
It can be fused to other effectors → RNase H1 or methylases for additional functions!
Let’s check out how they did it!
The Core Design Idea: Pseudo-Guide DNA
The author started with a simple question: how much RNA does Cas12 really need?
To find out, they trimmed the scaffold region (the stabilizing, non-targeting region of the crRNA) by 5, 10, 15 nt, and also made a version with no scaffold at all! Then they tested 8 different Cas12 enzymes.
Interesting results: only Cas12i1 and AsCas12a retained strong single-stranded DNA (ssDNA) cleavage with a no-scaffold RNA guide, and none worked against double-stranded DNA (dsDNA)! This suggests that these two enzymes have some unique conformational flexibility.
Is it enough to replace the RNA guide with a DNA guide?
Only one way to find out! The idea is straightforward: with an RNA guide, you target DNA. Can you use a DNA guide to target RNA? The authors tested a few guide formats against microRNAs:
With a “handle” (a scaffold-mimicking ssDNA loop) on the 5’ end
With a 3’ handle
Both 3’ and 5’ handle
No handle → just a dsDNA strand
The winner? The 3’-handle design, which they called ΨDNA. It seems to mimic the native crRNA scaffold, stabilizing the Cas12 complex and enabling RNA targeting!
Putting ΨDNA to Work: In Vitro…
The team started the tests in vitro.
ΨDNA activated Cas12-mediated collateral cleavage for microRNA detection. In particular, the DNA guide worked only when the correct miR-21, miR-122, and miR-155 were present. These are important microRNAs involved in cancer and hepatitis!
The researcher extended the assay to longer ~1.7 kb RNA targets made by in vitro transcription. AsCas12a performs especially well here, scoring 24/26 ΨDNAs (92.3%), while Cas12i1 scores 9/26 (34.6%). The system can discriminate mismatches, is robust, and doesn’t cut RNA directly!
So, why not test it for diagnostics?
The authors tested synthetic mimics of viral RNA from:
HCV
Dengue virus
Zika virus
For clinical applications, they focused on HCV. The team tested 40 clinical samples, with 20 positive and 20 negative. The system detected every positive sample, with 100% diagnostic accuracy!
So, ΨDNA can function as a real diagnostic RNA detection platform!
… And Inside Cells
But the coolest part happened inside cells.
The team co-transfected HEK293T cells with:
AsCas12a-GFP
mCherry reporter
and ΨDNAs targeting the mCherry mRNA.
The results? The mCherry fluorescence drops, and RNA sequencing shows that the mRNA levels also drop. How does it work? Probably via ribosome stalling, which leads to RNA degradation!
An interesting detail: ΨDNA alone reduces RNA expression, probably by simply binding to the complementary DNA. But still, AsCas12a significantly enhances repression! And the team didn’t stop here.
They wanted to knockdown endogenous genes. So, they targeted 3:
PPIA
RPL4
PCSK9
Across these targets, the team reported 50-70% reduction in mRNA relative to controls! And it worked in 3 other cell lines: HeLa, HepG2, and MCF-7.
Multiplexing, Dual Targeting, and Fusion Proteins
The researchers showed 3 “advanced” applications.
First, a multiplexed version. They designed different combinations targeting:
PPIA
RPL4
NRAS
PCSK9
All at once! They simply mixed different ΨDNAs, sent them into cells, and achieved simultaneous knockdown of multiple transcripts. How good is it, you ask? In the best cases, they see around 70-80% reduction! Not too shabby.
And they didn’t stop there.
They also showed that you can knockdown RNA and edit the genome simultaneously! Here’s how it works. You deliver:
crRNA to target CCR5 in DNA → standard genome editing
ΨDNA to target endogenous RNA → the new system
AsCas12a-GFP → to act on everything
What do they see? Well:
CCR5 indels at around 10-15% efficiency
Strong RNA knockdown
All at the same time! Just using a single AsCas12a, you get a permanent DNA edit and a transient RNA-level knockdown! This could be helpful in therapeutic settings, where you need both long-term genome editing and immediate RNA reduction.
Finally, they combined AsCas12a-ΨDNA with other proteins.
Fusing to RNase H1 enhanced RNA degradation, boosting the knockdown by an additional 15% over normal AsCas12a! Next, they fused AsCas12a-ΨDNA to METTL3, a methylase, showing that it can precisely edit RNA chemistry.
DNA Takes Over CRISPR
Such extensive work! Amazing.
The team extended the potential of Cas12 nucleases with ΨDNA. Now, they’re not limited to cutting DNA anymore! It becomes a versatile platform. But I’m excited by the practical advantages of DNA guides. Compared to RNA, they are:
cheaper
more stable
easier to make at scale
compatible with rapid iteration!
Sure, they can’t be produced directly inside cells, like RNA. So you can’t put it in a plasmid and forget about it. But for most transient applications, this isn’t a problem! And yes, I’m a DNA fan boy; what do you want?
The other main limitations are the classic ones with CRISPR-Cas:
Guide design, target accessibility, and Cas12 presence matter a lot. But at least with DNA it’s easier to iterate on guide design!
The knockdown mechanism is unclear. There’s ribosome stalling, RNase H1 involvement, and more.
But honestly, an amazing paper! With great applications in therapeutics, diagnostics, and metabolic control. Go and read it here. It’s very detailed, and I had to skip tons, but it’s worth a read!
If you made it this far, thank you! What do you think of ΨDNA? Do you think DNA has a place in the CRISPR-Cas world? Reply and let me know!
P.S: Know someone interested in genome editing? Share it with them!
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