How Type V CRISPR Is Revolutionizing Genetic Engineering
In the hidden world of bacterial immunity, scientists have discovered genetic scissors that are writing the future of medicine.
Imagine a pair of molecular scissors so precise it can edit a single misspelled letter among the 3 billion that make up the human genetic code. This is the promise of CRISPR-Cas gene editing, a technology adapted from nature's own defense system found in bacteria. Among the various CRISPR systems, one family—Type V—stands out for its astonishing diversity and special talents, from its compact size perfect for gene therapy to its unexpected origins in genetic "parasites."
When people hear "CRISPR," they often think of Cas9, the original "genetic scissors" that won the Nobel Prize in Chemistry in 2020. But Cas9 is just one of many CRISPR-associated proteins in nature's toolkit 2 .
CRISPR-Cas systems are broadly divided into two classes. Class 1 uses multi-protein complexes to target invaders, while Class 2 employs single proteins, making them ideal for genetic engineering 2 6 . Type V belongs to this more manageable Class 2, alongside Type II (which includes Cas9) and Type VI (which targets RNA) 6 .
| Type | Key Effectors | Target | PAM Preference | Key Features |
|---|---|---|---|---|
| Type II | Cas9 | DNA | G-rich (e.g., NGG) | First discovered, requires tracrRNA 6 |
| Type V | Cas12a, Cas12f, Cas12n | DNA | T-rich or A-rich | Single RuvC domain, often smaller size 5 6 |
| Type VI | Cas13 | RNA | None | Targets RNA, exhibits collateral cleavage 2 |
What truly sets Type V systems apart is their molecular architecture. Unlike Cas9, which uses two different nuclease domains to cut DNA, Type V effectors typically rely on just one catalytic domain (RuvC) to cut both strands of DNA 6 . This simpler, often more compact design has made them particularly valuable for therapeutic applications where size matters.
In a fascinating twist of evolution, Type V CRISPR systems appear to have originated from transposon-encoded TnpB nucleases—essentially genetic parasites that live within bacterial genomes 1 . The recent discovery of TranCs (transposon-CRISPR intermediates) has provided the missing link in this evolutionary journey 1 .
Single-molecule reRNA/omega RNA
Transposon activity
Associated with transposons
Both reRNAs and CRISPR RNAs
Transitional immune function
Intermediate organizations
Split crRNA and tracrRNA
Adaptive immunity
Dedicated CRISPR arrays
The key transition occurred through "functional RNA splitting" 1 . While TnpB utilizes a single-molecule guide RNA called right-end RNA (reRNA) or omega RNA, Type V systems employ a split system with separate CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) molecules 1 . This crucial splitting event allowed the system to evolve from transposon function to adaptive immunity, ultimately giving rise to the diverse Type V CRISPR-Cas systems we see today 1 .
The Type V family is remarkably diverse, with various members specializing in different functions:
The first discovered Type V effector, known for creating staggered DNA cuts with 5' overhangs rather than the blunt ends produced by Cas9 6 .
A recently characterized member that uniquely recognizes rare A-rich PAM sequences, significantly expanding the targeting range of CRISPR technologies 5 .
The compact size of many Type V effectors is particularly valuable for gene therapy. For instance, Cas12f-based editors are small enough to be packaged into adeno-associated viruses (AAVs)—the leading delivery vehicle for gene therapies—overcoming a major limitation of bulkier Cas proteins 7 .
The 2025 study "Functional RNA splitting drove the evolutionary emergence of type V CRISPR-Cas systems from transposons" represents a watershed moment in our understanding of CRISPR evolution 1 . The international team of scientists set out to solve a fundamental mystery: how did transposon-encoded TnpB nucleases evolve into the sophisticated immune systems we know as Type V CRISPR?
They discovered TranCs (transposon-CRISPR intermediates) derived from distinct IS605- or IS607-TnpB lineages that could utilize both CRISPR RNAs and the ancestral reRNAs to direct DNA cleavage 1 .
Using cryo-electron microscopy (cryo-EM), they determined the high-resolution structure of LaTranC from Lawsonibacter sp., revealing a striking resemblance to the ISDra2 TnpB complex but with a critical difference—the guide RNA was functionally split into tracrRNA and crRNA 1 .
To confirm their hypothesis, they engineered an RNA split version of ISDra2 TnpB and demonstrated that this modified system could function with a CRISPR array, effectively recreating the key evolutionary step in the laboratory 1 .
The findings were profound. The structural data showed that despite their similarity, LaTranC's guide RNA was functionally split into separate tracrRNA and crRNA molecules, unlike the single-molecule reRNA used by TnpB 1 . Even more compelling, the engineered RNA split of ISDra2 TnpB successfully enabled activity with a CRISPR array 1 .
This research demonstrates that functional RNA splitting was likely the primary molecular event driving the emergence of diverse Type V CRISPR-Cas systems from their transposon ancestors 1 . The implications extend beyond evolutionary biology—understanding these natural engineering principles helps scientists design better gene-editing tools.
| Reagent/Tool | Function | Example Applications |
|---|---|---|
| Cryo-EM | Determines high-resolution 3D structures of molecular complexes | Elucidating Cas12n mechanism and PAM recognition 5 |
| Lipid Nanoparticles (LNPs) | Delivery vehicles for in vivo CRISPR therapies | Successful systemic delivery in clinical trials 4 |
| Cas12n sgRNA | Engineered single-guide RNA combining crRNA and tracrRNA | Converting RdCas12n into effective genome editor in human cells 5 |
| Pro-CRISPR Factors (Pcr) | Non-Cas accessory genes enhancing CRISPR function | Improving efficiency and adding functionalities to CRISPR systems 8 |
| Protein Language Models | AI systems generating novel CRISPR proteins | Designing OpenCRISPR-1 with optimal editing properties 3 |
Type V systems are already making the leap from basic research to clinical applications. Intellia Therapeutics has reported remarkable success with an in vivo CRISPR therapy for hereditary transthyretin amyloidosis (hATTR) 4 . Using lipid nanoparticles to deliver Type V components, they achieved ~90% reduction in disease-causing protein levels that remained stable over two years 4 .
The advantages of Type V systems are particularly evident in these clinical applications. Their often smaller size allows for easier packaging into delivery vehicles, while their different PAM preferences expand the targetable genetic space. Furthermore, the staggered cuts produced by some Type V effectors can facilitate more precise genetic insertions 6 .
As we look ahead, the convergence of AI with CRISPR research promises to accelerate discoveries. Researchers have already used large language models to generate artificial CRISPR proteins like OpenCRISPR-1, which shows comparable or improved activity relative to natural Cas9 despite being 400 mutations away in sequence 3 . This AI-driven approach has expanded protein diversity by 4.8-fold compared to natural CRISPR-Cas systems 3 .
The journey of Type V CRISPR—from genetic parasites to precision medicine tools—exemplifies how understanding fundamental biology can unlock transformative technologies. As we continue to unravel the mysteries of these molecular scissors, we move closer to a future where genetic diseases become manageable, sustainable agriculture flourishes, and our ability to rewrite the code of life is limited only by our imagination.