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| Effect of Different DNA Activator Structures on the?trans-Cleavage Activity of Cas12a |
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| DOI： |
| KeyWord:Cas12a ?protospacer adjacent motif（PAM） ?dsDNA activator ?ssDNA activator ?in vitro biosensing |
| Author | Institution |
| FEI Xin-rui，LIU Xiao-ling，LIU Cheng-hui |
Key Laboratory of Applied Surface and Colloid Chemistry，Ministry of Education;Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province，School of Chemistry & Chemical Engineering，Shaanxi Normal University， Xi′an ，China |
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| 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． |
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