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不同结构的DNA激活序列对Cas12a反式切割活性的影响
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费心蕊,刘晓玲,刘成辉 应用表面与胶体化学教育部重点实验室陕西省生命科学分析化学重点实验室陕西师范大学 化学化工学院陕西 西安 710119 
基金项目:the National Natural Science Foundation of China(22104077);the Fundamental Research Funds for the Central Universities(GK202101001,GK202103044);the Natural Science Foundation of Shaanxi Province(2021TD-42,2020JQ-407)
中文摘要: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。
中文关键词:Cas12a  ?前间区序列邻近基序(PAM)  ?双链DNA激活序列  ?单链DNA激活序列  ?体外传感
 
Effect of Different DNA Activator Structures on the?trans-Cleavage Activity of Cas12a
Abstract: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(ssDNA) activators with varying structures are employed in the existing literatures,and there still lacks systematic guiding principles for Cas12a activator design.Herein,the impact of DNA activator structures on the?trans-cleavage activity 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 design 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 reduce 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 activators 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 PAM-containing dsDNA activator.The results presented in this study may help the fabrication of high-efficient CRISPR-Cas12a-based in vitro biosensing systems.
Key Words:Cas12a  ?protospacer adjacent motif(PAM)  ?dsDNA activator  ?ssDNA activator  ?in vitro biosensing
引用本文:费心蕊,刘晓玲,刘成辉.不同结构的DNA激活序列对Cas12a反式切割活性的影响[J].分析测试学报,2022,41(4):610-617.
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