Gene-editing tools, which function like molecular scissors, are derived from the immune systems of bacteria. Within this family of tools, known as CRISPR-Cas, the Cas12a protein is a notable example. When paired with its guide, a molecule called CRISPR RNA (crRNA), Cas12a becomes a programmable system for finding and altering specific DNA sequences.
The Cas12a-crRNA Targeting Complex
The functional core of this tool is the Cas12a-crRNA ribonucleoprotein (RNP) complex. The Cas12a protein is a nuclease that acts as the cutting component, but it is inactive on its own. To gain its function, it must bind with a crRNA molecule, which provides the system’s specificity.
The crRNA molecule has two distinct parts. The first is a constant structural scaffold called the direct repeat, which the Cas12a protein recognizes and binds. The second is a customizable spacer region of 20-24 nucleotides, designed to be complementary to a target DNA sequence. When joined, they form an active RNP complex programmed to scan the genome for a DNA sequence that matches the crRNA’s spacer region.
Mechanism of On-Target DNA Cleavage
The complex’s search begins by looking for a short sequence known as a Protospacer Adjacent Motif (PAM), which acts as a docking site. For Cas12a, the PAM is a T-rich sequence like TTTN, and its recognition is required for the complex to engage with the DNA.
After binding to the PAM, Cas12a unwinds the adjacent DNA double helix. This separation allows the crRNA’s spacer to check for a match with one of the DNA strands, called the target strand. If the sequences are complementary, the crRNA binds to it, creating a stable R-loop structure and displacing the non-target strand.
This successful binding event triggers a conformational change in the Cas12a protein, activating its RuvC nuclease domain. Cas12a then proceeds to cut both strands of the target DNA, severing the molecule.
Unique Properties of the Cas12a System
The Cas12a system has several characteristics that set it apart from other CRISPR tools like Cas9. First, it recognizes a T-rich PAM (e.g., 5′-TTTV-3′), which differs from the G-rich PAM required by SpCas9. This expands the range of genomic sites that can be targeted.
Another difference is the type of DNA cut it produces. Cas12a makes a “staggered” cut instead of a “blunt” one, cleaving each strand at a different position. This process results in short, single-stranded overhangs on the cut DNA, which is useful for techniques involving the insertion of new DNA.
The system is also simpler in its RNA requirements. While Cas9 needs two RNA molecules (crRNA and tracrRNA), Cas12a only needs a single crRNA. Cas12a can also process its own precursor crRNA into the mature guide molecule, which simplifies its use in research.
The Collateral Cleavage Effect
Beyond its precise on-target cutting, Cas12a has a secondary activity. After the complex binds to and cleaves its intended DNA target, the protein enters a hyperactive state. In this state, Cas12a’s nuclease domain indiscriminately cleaves any single-stranded DNA (ssDNA) molecules nearby.
This non-specific phenomenon is known as collateral or trans-cleavage. The protein does not require a matching sequence for this secondary cutting. The initial on-target event acts as the trigger, and the collateral activity continues as long as the complex is bound to its target.
Applications in Biotechnology and Diagnostics
The activities of the Cas12a system have been adapted for applications in science and medicine. Its primary on-target DNA cleavage is used for gene editing. Researchers introduce precise breaks in the DNA of cells, plants, and animals to study gene function or for therapeutic purposes, such as correcting mutations that cause inherited diseases.
The secondary collateral cleavage effect has been harnessed for molecular diagnostics. Diagnostic tests are designed by adding fluorescent reporter molecules, which are short pieces of ssDNA, to a sample along with the programmed Cas12a complex. If the target DNA from a pathogen is present, the complex will find and cut it, activating the collateral cleavage and causing Cas12a to shred the reporter molecules. The destruction of these reporters releases a measurable fluorescent signal, indicating the pathogen’s presence.