Zhang Wei, Zhong Yinyin, Wang Jiaqi, Zou Guangrong, Chen Qiaozhen, Liu Chaoxing
Guangdong Provincial Key Laboratory of Digestive Cancer Research, Digestive Diseases Center, Scientific Research Center, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen, Guangdong 518107, P.R. China.
Department of Clinical Laboratory, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen, Guangdong 518107, P.R. China.
Nucleic Acids Res. 2025 Jan 24;53(3). doi: 10.1093/nar/gkaf040.
CRISPR-Cas12a technology has transformative potential, but as its applications grow, enhancing its inherent functionalities is essential to meet diverse demands. Here, we reveal a regulatory mechanism for LbCas12a through direct repeat (DR) region 3' end modifications and de-modifications, which can regulate LbCas12a's cis- and trans-cleavage activities. We extensively explored the effects of introducing phosphorylation, DNA, photo-cleavable linker, DNA modifications at the DR 3' end on LbCas12a's functionality. We find that the temporary inhibitory function of Cas12a can be reactivated by DR 3' end modification corresponding substances, such as alkaline phosphatase (ALP), immunoglobulin G (IgG), alpha-fetoprotein (AFP), DNA exonucleases, ultraviolet radiation, and DNA glycosylases, which greatly expand the scope of application of Cas12a. Clinical applications demonstrated promising results in ALP, AFP, and trace Epstein-Barr virus detection compared to gold standard methods. Our research provides valuable insights into regulating LbCas12a activity through direct modification of DR and significantly expands its potential clinical detection targets, paving the way for future universal clustered regularly interspaced short palindromic repeats (CRISPR) diagnostic strategies.
CRISPR-Cas12a技术具有变革潜力，但随着其应用的增加，增强其固有功能对于满足多样化需求至关重要。在这里，我们通过直接重复（DR）区域3'末端修饰和去修饰揭示了一种LbCas12a的调控机制，该机制可以调节LbCas12a的顺式和反式切割活性。我们广泛探索了在DR 3'末端引入磷酸化、DNA、光可裂解接头、DNA修饰对LbCas12a功能的影响。我们发现，Cas12a的临时抑制功能可以被DR 3'末端修饰相应物质重新激活，如碱性磷酸酶（ALP）、免疫球蛋白G（IgG）、甲胎蛋白（AFP）、DNA外切核酸酶、紫外线辐射和DNA糖基化酶，这大大扩展了Cas12a的应用范围。与金标准方法相比，临床应用在ALP、AFP和微量爱泼斯坦-巴尔病毒检测中显示出有前景的结果。我们的研究为通过直接修饰DR来调节LbCas12a活性提供了有价值的见解，并显著扩展了其潜在的临床检测靶点，为未来通用的成簇规律间隔短回文重复序列（CRISPR）诊断策略铺平了道路。
