The CRISPR-Cas systems are adaptive immune mechanisms found in prokaryotes, primarily bacteria and archaea, which protect against invading genetic elements such as phages and plasmids. These systems have been harnessed for various biotechnological applications, including genome editing and molecular diagnostics. Cas12a2, a particular variant within the CRISPR-Cas family, has garnered interest due to its ability to cleave single-stranded nucleic acids upon activation by a specific RNA target. This unique property makes it a promising candidate for developing sensitive and specific diagnostic tools. However, the challenge lies in fine-tuning its activity to ensure high specificity for single-stranded nucleic acids while minimizing off-target effects on double-stranded nucleic acids, which is crucial for accurate diagnostics.
Current approaches to utilizing Cas12a2 in diagnostics face significant limitations due to its inherent non-specific cleavage activity, which can lead to unintended degradation of both single-stranded and double-stranded nucleic acids. This collateral damage can result in false positives and negatives, reducing the reliability and accuracy of diagnostic tests. Additionally, the molecular basis of Cas12a2’s activation and substrate specificity remains poorly understood, complicating efforts to engineer the enzyme for improved performance. Existing methods also struggle with the enzyme’s sensitivity to mismatches in the target RNA sequence, further affecting its diagnostic utility. Therefore, there is a pressing need for modified versions of Cas12a2 that can selectively cleave single-stranded nucleic acids while sparing double-stranded DNA, thereby enhancing the specificity and effectiveness of CRISPR-based diagnostic assays.
The technology involves engineered Cas12a2 molecules that are designed to selectively cleave single-stranded nucleic acids while minimizing their activity on double-stranded nucleic acids. This selectivity is achieved through specific mutations in the Cas12a2 protein, such as Y465A and Y1080A, which reduce the cleavage of double-stranded DNA. Another mutation, Y1069A, enables the enzyme to cleave single-stranded DNA but not single-stranded RNA or double-stranded DNA. The Cas12a2 structure includes a recognition lobe and a nuclease lobe, with the active site exposed upon binding to a target RNA sequence. The binding induces conformational changes that create a positively charged groove, accommodating single-stranded nucleic acids for cleavage. These modifications make Cas12a2 suitable for diagnostic tools to detect specific RNA sequences, providing a measurable signal in the presence of target RNA while reducing off-target effects on double-stranded DNA.
What differentiates this technology is its precise control over nucleic acid cleavage, which is achieved by altering specific residues within the Cas12a2 protein. Unlike other CRISPR systems that may have broader or less specific cleavage activities, the modified Cas12a2 molecules exhibit a highly selective cleavage pattern. This selectivity is crucial for diagnostic applications where accuracy is paramount. The ability to tune the enzyme’s activity to target only specific nucleic acids while minimizing off-target effects enhances its utility in sensitive and specific detection of RNA sequences. Additionally, the structural insights into how the Cas12a2 undergoes conformational changes upon RNA target binding provide a deeper understanding of its mechanism, further differentiating it from other nucleases and making it a versatile tool in molecular diagnostics.