Journal
FRONTIERS IN MICROBIOLOGY
Volume 12, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fmicb.2021.766464
Keywords
CRISPR diagnostics; trans-cleavage; enzymatic units; Cas12; transU
Categories
Funding
- National Natural Science Foundation of China [31922046, 31770057]
- Sanming Project of Medicine in Shenzhen [SZSM202011017]
- National Key Research and Development Program of China [2018YFA0903700, 2018YFC0809200]
- Guangdong Science and Technology Foundation [2020B1111160001, B2019228, 2020A1515110744]
- Shenzhen Science and Technology Foundation [SGLH20180625171602058, 201906133000069, GJHZ20200731095604013, JCYJ20210324102803009]
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Despite the great potential of CRISPR-Dx technology in diagnostic sensitivity, specificity, convenience, and portability, the lack of standardized definition of Cas trans-cleavage enzymatic units has impeded the development of the industry. By optimizing the reaction systems for Cas12a and defining its trans-cleavage units (transU), researchers can now have a reference point for standardization and further research in the field.
The CRISPR diagnostic (CRISPR-Dx) technology that employs the trans-cleavage activities has shown great potential in diagnostic sensitivity, specificity, convenience, and portability, and has been recognized as the next-generation diagnostic methods. However, due to the lack of standardized definition of Cas trans-cleavage enzymatic units, it is difficult to standardize the present CRISPR-Dx systems, which have undoubtedly impeded the development of the CRISPR-Dx industry. To solve the problem, we here first systematically optimized the reaction systems for Cas12a, and then defined its trans-cleavage units (transU), which we believe will be of great importance and interest to researchers in both molecular diagnostic industry and basic research. Moreover, a simple protocol was provided to facilitate a step-by-step measurement of the Cas12a transU, which can also act as a reference for the definition of the transU for other Cas proteins.
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