Part:BBa_K2323010
Lwa Cas13a coding sequence
Cas13a is a CRISPR/Cas effector that specifically binds and digests RNA based on its crRNA sequence. After digestion of this target sequence, Cas13a undergoes conformation change and presents collateral activity, i.e. it indiscriminately digests RNA molecules.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1286
Illegal BglII site found at 2966 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2365
Literature Survey: Contribution by Team IISER_Berhampur 2021
Group: Team IISER_Berhampur 2021
Summary: We learned from literature that Cas13a protein designed by Team iGEM Munich in iGEM 2017 from Leptotrichia wadei (LwaCas13a) for in vitro systems can be engineered for in vivo use to target endogenous transcripts (RNA targeting) in human HEK293FT cells as well as in plants. Moreover, the monomeric superfolder GFP (msfGFP) can be fused to LwaCas13a that can provide more stability and specificity. Also, it can be developed for programmable tracking of transcripts in live cells.
Methodology
We propose a targeted RNA knockdown using a mammalian codon-optimized Cas13a vector LwaCas13a-msfGFP-NLS from - Addgene Plasmid #91902 Additionally, to evaluate the Cas13a activity we propose to design single guide RNAs against two fluorescence (preferably using luciferase) reporters, where one of them serves as Cas13a target and the other serves as a control. Upon transfecting them into mammalian HEK293FT cells for 48 hours, we would evaluate the fluorescence activity in the cells. The study from [Ref. 1] shows that LwaCas13a is a useful tool for mammalian knockdown experiments. This can be also implemented in plant cells with a similar approach. It also shows that if this vector can be engineered for targeting different genes, it can give varying, but better levels of RNA knockdown as RNAi and its efficiency is transcript dependent.
The above-mentioned methodology can be tested with the in-detail protocols from [Ref. 1]
Below is a concise comparison between different Cas13a orthologs including LwaCas13a (adapted from [Ref. 2])
Conclusion
Although LwaCas13a is an excellent tool for in vitro RNA digestion, as conceptualized by iGEM Munich 2017, it can be also used for RNA knockdown in mammalian cells. Also, the knockdown efficiency mainly depends on three factors:1) the type of ortholog, 2) the system: mammalian/ bacterial/ in vitro, 3) the target gene.
References
1) Abudayyeh, O. O., Gootenberg, J. S., Essletzbichler, P., Han, S., Joung, J., Belanto, J. J., Verdine, V., Cox, D. B., Kellner, M. J., Regev, A., Lander, E. S., Voytas, D. F., Ting, A. Y., & Zhang, F. (2017). RNA targeting with CRISPR–CAS13. Nature, 550(7675), 280–284. https://doi.org/10.1038/nature24049
2) Cas13. Zhang Lab. (n.d.). Retrieved October 7, 2021, from https://zlab.bio/cas13.
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