Cas13b is a component of the CRISPR-Cas molecular system and part of a family of proteins. It is an enzyme that can be programmed to locate and interact with specific sequences of genetic material inside a cell. This capability is derived from its natural function as part of the immune system in bacteria and archaea, where it helps defend against invading viruses.
Researchers have harnessed this function, developing Cas13b into a programmable tool for various uses in biotechnology and medicine. Its structure, composed of around 1,100 to 1,200 amino acids, enables it to be a precise tool for genetic applications.
The RNA-Targeting Mechanism of Cas13b
The primary function of the Cas13b protein is to target and cut molecules of ribonucleic acid, or RNA, which distinguishes it from other tools that target DNA. The system operates using a guide RNA (gRNA), a small, designed RNA molecule containing a sequence complementary to the intended target. The Cas13b protein binds to this gRNA, forming a complex that searches the cell for RNA molecules matching the gRNA’s sequence.
Once the Cas13b-gRNA complex locates and binds to the correct RNA strand, the enzyme becomes activated. In its active state, Cas13b uses two distinct catalytic centers to cleave the single-stranded RNA target. This action destroys the target RNA molecule, preventing it from carrying out its function.
A unique feature of Cas13b’s mechanism is a phenomenon known as “collateral cleavage.” After the enzyme has cut its intended RNA target, it does not become inactive. Instead, the activated Cas13b protein begins to cut other, non-targeted RNA molecules in its immediate vicinity. Scientists have found ways to harness this specific property for certain applications.
Key Distinctions from Cas9
While Cas13b is part of the broader CRISPR family, it has several distinctions from the more widely known Cas9 protein. The most fundamental difference is the type of genetic material each enzyme targets. Cas13b is an RNase designed to find and cut RNA, while Cas9 is a DNase that targets and cuts DNA, the cell’s primary genetic blueprint.
This difference in targets leads to a divergence in the permanence of their effects. Because Cas9 alters the DNA sequence of a genome, its edits are permanent and passed down through cell division. Cas13b’s action of degrading RNA is temporary, as RNA molecules are constantly being produced and degraded by the cell. The effect lasts only as long as the Cas13b tool is active, without changing the underlying genetic code.
Another point of contrast is the collateral cleavage activity inherent to Cas13b, which Cas9 does not exhibit. Cas9 makes a precise cut at its DNA target and then disengages. Finally, their targeting requirements differ. Cas9 needs a specific DNA sequence known as a protospacer adjacent motif (PAM) next to its target site. Cas13b has a less stringent requirement for a similar sequence, called a protospacer flanking site (PFS), making it more flexible in the RNA sequences it can target.
Applications in Diagnostics
The collateral cleavage activity of Cas13b has been adapted for use in diagnostic tools. This feature is the foundation for highly sensitive detection platforms that produce a clear signal when a specific RNA sequence is present in a sample.
In these diagnostic assays, a patient sample is mixed with the Cas13b-gRNA complex, which is programmed to find a specific viral or bacterial RNA. Also included are reporter molecules, which consist of a short RNA strand with a fluorescent or color-producing molecule at one end and a quencher molecule at the other that keeps the signal off.
If the target RNA is present, Cas13b becomes activated and its collateral cleavage function begins cutting any RNA in the vicinity, including the reporters. As the reporters are cut, the fluorescent or color-producing component is separated from the quencher, releasing a detectable signal. This process forms the basis of diagnostic systems like SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing), allowing for the rapid and specific detection of nucleic acids.
Therapeutic and Research Capabilities
Beyond diagnostics, Cas13b has a range of capabilities for therapeutic intervention and research. One application is RNA knockdown, a method to temporarily silence a specific gene. By designing a gRNA that targets a particular messenger RNA (mRNA), Cas13b can destroy that mRNA, preventing the cell from producing a potentially harmful protein without altering the genetic code.
The enzyme also holds potential as a direct-acting antiviral agent. Many viruses, including influenza and coronaviruses, have RNA genomes. Cas13b can be programmed to recognize and destroy the viral RNA genome inside an infected cell, which would halt the virus’s ability to replicate and spread.
Furthermore, Cas13b can be modified for precise RNA editing. Scientists have engineered a “deactivated” version of the enzyme (dCas13b) that has its cutting function disabled. This dCas13b can still be guided to a specific RNA sequence, but it only binds to it. By fusing other functional proteins to dCas13b, such as enzymes that can change one RNA base to another, researchers can perform precise edits to RNA transcripts, offering a therapeutic strategy that avoids permanent changes to DNA.