Enhancement of CRISPR-Cas12a System through Universal Circular RNA Design

Abstract

The combination of isothermal amplification and CRISPR-Cas12a system is a valuable tool for detecting nucleic acids, especially in emergency situations or disease outbreaks. However, achieving compatibility between these technologies has been a challenge because the CRISPR reaction eagerly cleaves nucleic acid amplification template before isothermal amplification is complete. This study presents a method called CIRCLE, which effectively addresses the compatibility issue by dividing crRNA into a universal circular DR region with a PC linker and a replaceable spacer region. Different targets could be detected in a single separate way by changing the spacer region. Furthermore, DNA modifications were firstly introduced into circular crRNA to regulate CRISPR-Cas12a system using base excision repair (BER) enzymes. The programmable strategy showed potential to detect different BER enzymes and regulate Cas12a gene-editing activity by adjusting BER enzyme levels. In the future, it holds promise for selectively editing genes in cells that express high levels of specific BER enzymes, such as UDG, in colon cancer cells. The findings of this study contribute to advancing the field of nucleic acid detection and gene editing. The CIRCLE method is a programmable and resource efficient approach for medical diagnostic applications, making it particularly valuable in future clinical settings.