Part:BBa_K4170013
LbuCas13a with 6XHis purification tag
This plasmid contains the coding sequence (CDS) of codon-optimized LbuCas13a protein for recombinant LbuCas13a protein expression in E.coli in fusion with the 6xHis affinity tag at the N-terminus of the Cas13a Lbu (BBa_K2926001) to facilitate the efficient purification of the protein utilizing a Ni-NTA affinity purification methodology. for efficient protein purification utilizing.The LbuCas13a initial coding sequence derives from the genus Leptotrichia and then appropriate optimization strategies were applied.
This plasmid has been constructed from pGJK_His-SUMO-LbuCas13a plasmid, deposited by Jennifer Doudna and her colleagues at Addgene plasmid repository, after successive mutagenesis PCR and cloning steps to remove the restrictions sites that are incompatible with RFC[10] biobrick standards. After multiple mutagenesis PCRs, the final assembly of the PCR amplified products was ligated with the linearized pSB1C3 plasmid backbone utilizing a Golden Gate-based SevaBrick assembly method introduced by Stamatios G. Damalas and colleagues. The final LbuCas13a coding sequence lacks of the 5 initial restriction sites (Enzyme: position in plasmid, XbaI:5030, SapI: 6263, EcorI:6987 & 8263, BsaI: 8339) ensuring the biobrick RFC [10] compatibility.
Usage and biology
CRISPR/Cas13a systems as RNA sensors
CRISPR-Cas systems are RNA-guided adaptive immune systems that protect prokaryotes from foreign genetic elements derived from evading viruses and phages (Shan et al., 2019). The Cas13a is ribonuclease with a double biological functionality: catalyzes the crRNA maturation and degrades the RNA-guided ssRNA (single-stranded RNA) interdependently using two separated catalytic sites (Rath et al., 2015). Generally, CRISPR/Cas system can be divided into two main classes, class I and II, according to the system comprising a single or multiple effectors (Liu et al., 2017). Among them, class II (e.g., Cas9, Cas12, and Cas13) possesses more widespread application, due to its simple components (a single effector protein and a programmable guide RNA, Wang et al., 2021). Cas13 can be further divided into four subtypes, Cas13a-d, exhibiting diverse primary sequences except the two highly conserved HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domains, which are responsible for both cis- and trans-RNase activities (Florczuk et al., 2017). Structural studies revealed that Cas13a adopts a bilobed architecture including recognition (REC) and nuclease (NUC) lobes (Wang et al., 2021, Zhou et al., 2020). CRISPR-Cas12,13,14 exhibits nonspecific degradation of non target (trans cleavage) after specific recognition of nucleic acids, thus CRISPR/Cas biology promises rapid, accurate, and portable diagnostic tools, the next-generation diagnostics. Cas13a due to its propensity to cleave RNAs after binding a user-defined RNA target sequence, is used to detect single molecules of RNA species with high specificity (Liu et al., 2017).
Similarly to Cas9, Cas13a recognizes the short hairpin of the crRNA and made a complex. The target specificity is encoded by the 28-30-nt crRNA spacer sequence which is complementary to the target region. Cas13s exhibit collateral activity and after recognition and cleavage of the target transcript, degrades non-specifically any nearby RNAs regardless of complementarity to the spacer.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3189
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1267
- 1000COMPATIBLE WITH RFC[1000]
Citations
1. Kaminski, M., Abudayyeh, O., Gootenberg, J., Zhang, F. and Collins, J., (2021) "CRISPR-based diagnostics." Nature Biomedical Engineering, 5(7), pp.643-656.
2. Liu, L., Li, X., Ma, J., Li, Z., You, L., Wang, J., Wang, M., Zhang, X.and Wang, Y., (2017) "The Molecular Architecture for RNA - Guided RNA Cleavage by Cas13a." Cell, 170(4), pp .714 - 726.e10.2.
3. Rath, D., Amlinger, L., Rath, A. and Lundgren, M., (2015) "The CRISPR-Cas immune system: Biology, mechanisms and applications." Biochimie, 117, pp.119-128.
4. Shan, Y., Zhou, X., Huang, R.and Xing, D., (2019) "High - Fidelity and Rapid Quantification of miRNA Combining crRNA Programmability and CRISPR / Cas13a trans - Cleavage Activity." Analytical Chemistry, 91(8), pp .5278 - 5285. 3.
5. Wang, B., Zhang, T., Yin, J., Yu, Y., Xu, W., Ding, J., Patel, D. and Yang, H., (2021) "Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems." Molecular Cell, 81(5), pp.1100-1115.e5
6. Zhang, C., Konermann, S., Brideau, N., Lotfy, P., Wu, X., Novick, S., Strutzenberg, T., Griffin, P., Hsu, P.and Lyumkis, D., (2018) "Structural Basis for the RNA - Guided Ribonuclease Activity of CRISPR - Cas13d." Cell, 175(1), pp .212 - 223.e17.
| None |