Part:BBa_K4778002
Cas12a_sgRNA_D
CRISPR-Cas system is a natural immune system in bacteria and archaea, which is used to resist the invasion of foreign DNA. It protects bacteria from viruses and other exogenous DNA by identifying foreign DNA sequences and cleaving them into fragments. The core component of the CRISPR-Cas system is the Cas protein, which can recognize and cleave DNA.
Cas12a, also known as Cpf1, has a molecular weight of about 160kD. It is a kind of endonuclease that specifically cleaves assistant DNA (requires PAM site) under the guidance of sgRNA and trans-cleaves ssDNA after activation. As described by iGEM18_TJU_China, Cas12a’s cleavage activity can cleave the downstream sites of PAM sequence, and has strong trans-cleavage activity, so Cas12a can be used for signal conversion.
Biology
Fig1，The pattern diagram of Cas12a domains
The WED domain of Cas12a is responsible for combining with the stem-loop structure of sgRNA. The RuvC domain and Nuc domain undertake the work of nuclease sites.
Compared with Cas proteins in other CRISPR-Cas systems, Cas12a has some unique characteristics. First of all, Cas12a can be active in a wide temperature range, which makes it work in a variety of environmental conditions. Second, Cas12a has a smaller protein structure, which makes it more convenient for transport and application in vivo. Third, Cas12a also has high DNA cleaving efficiency and considerable non-specific cleaving tendency (trans-cleaving), which makes it have high application potential in the field of gene editing.
Usage
The work of Shi-YuanLi et al has proved that activated Cas12a has good trans-cleavage activity for ssDNA [1]. And this trans-cleavage activity is very efficient both inside and outside the cell. At the same time, this Cas protein has some unique advantages compared with other proteins of the same family.
Results
We take inspiration from Professor Nie Zhou's CONAN system [2] and continue to build and optimize our team's DRJ system. First of all, the ability of Cas12a trans-cleaving to release fluorescence group was verified. It is found that the cleaving efficiency of Cas12a can meet our need properly.
After continuous iteration, our system relies on Cas12a and the interaction between Cas12a, sgRNA_D and assistant DNA to amplify and detect trace nucleic acid signals.
References
[1]Li SY, Cheng QX, Liu JK, Nie XQ, Zhao GP, Wang J. CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA. Cell Res. 2018;28(4):491-493.
[2]Shi K, Xie S, Tian R, et al. A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics. Sci Adv. 2021;7(5):eabc7802. Published 2021 Jan 27.
[3]Swarts DC, van der Oost J, Jinek M. Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a. Mol Cell. 2017;66(2):221-233.e4. Sequence and Features
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