不同结构的DNA激活序列对Cas12a反式切割活性的影响

Effect of Different DNA Activator Structures on the trans-Cleavage Activity of Cas12a

CRISPR-Cas12a系统的反式切割活性在其识别特定的DNA激活序列后被激活,这不仅能实现特定DNA靶标的直接定量分析,同时也为构建针对多种生物标志物的体外传感体系带来了新的思路。然而,已有文献中所采用的双链DNA(dsDNA)和单链DNA(ssDNA)激活序列结构多种多样,缺乏全面、系统的设计指导原则。针对该问题,该文系统研究了不同结构的DNA激活序列对LbaCas12a反式切割活性的影响。通过对比研究,得出以下结论:(1)前间区序列邻近基序(PAM)位点有助于LbaCas12a更高效地靶向结合dsDNA激活序列和ssDNA激活序列;(2)PAM近端区域缺少序列片段会降低Cas12a-crRNA定位激活序列的效率;(3)删除PAM远端序列片段有利于增强LbaCas12a的反式切割活性;(4)由于省略了dsDNA解链过程,ssDNA激活序列在激活LbaCas12a的反式切割活性方面普遍比dsDNA激活序列产生的效果更好。根据这些发现,该文提出了一种LbaCas12a所青睐的高效激活序列结构,其激活的LbaCas2a反式酶切活性较采用含PAM位点的标准dsDNA激活序列高出3.7倍。研究结果为构建基于CRISPR-Cas12a的高效体外生物传感系统提供了重要支撑。

The trans-cleavage activity of CRISPR-Cas12a system could be activated after it recognizes the specific DNA activator sequences,which paves the foundation not only for direct target DNA detection but also for expanding indirect biosensing of various biomolecules.However,both double-strand DNA(dsDNA)and single-strand DNA(ss⁃DNA)activators with varying structures are employed in the existing literatures,and there still lacks systematic guid⁃ing principles for Cas12a activator design.Herein,the impact of DNA activator structures on the trans-cleavage ac⁃tivity of LbaCas12a is systematically studied by monitoring the fluorescence signal generated under the guidance of the same crRNA.According to a series of comparison,the following conclusions have been drawn for the sequence de⁃sign of DNA activators.(1)protospacer-adjacent motif(PAM)site helps LbaCas12a to target the dsDNA activators and the ssDNA activators with higher efficiency.(2)lacking the sequence fragment in the proximal region of PAM will re⁃duce the efficiency of Cas12a-crRNA in positioning the activator.(3)pre-deletion of the fragment adjacent to the crRNA-pairing sequence in the PAM-distal end is conducive to the LbaCas12a trans-cleavage activity.(4)ssDNA activators generally have better performance in activating the trans-cleavage activity of LbaCas12a than dsDNA activa⁃tors due to the omission of dsDNA unzipping.According to these findings,an efficient LbaCas12a-preferred activator structure is recommended,which could yield 3.7 times higher fluorescence intensity than the widely applied PAMcontaining dsDNA activator.The results presented in this study may help the fabrication of high-efficient CRISPRCas12a-based in vitro biosensing systems.