Composite

Part:BBa_K4139007:Design

Designed by: Emily Hagens   Group: iGEM22_Lethbridge   (2022-10-11)


mcyH crRNA for Lwa Cas13a (for E.coli, Composite)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 85
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

In order to determine which region of the mcyH gene our crRNA should target, we used the RNAfold tool. This allowed us to locate a target sequence that met the ideal design criteria of being close to the 5' end of the sequence, being relatively unfolded region in the mRNA secondary structure, and having a high probability of remaining single-stranded.

This crRNA coding region was designed to be the reverse compliment of the region it will target.

Note that this crRNA will target E.coli that has been transformed with the codon-optimized mcyH gene, rather than the mcyH gene from the Microcystis aeruginosa genome.


Source

This part was designed using a segment of the mcyH gene as a reference (GenBank: AAF00956.1)

References

Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 2016 Aug 5;353(6299):aaf5573. doi: 10.1126/science.aaf5573. Epub 2016 Jun 2. PMID: 27256883; PMCID: PMC5127784.

Gruber AR, Lorenz R, Bernhart SH, Neuböck R, Hofacker IL. The Vienna RNA Websuite. Nucleic Acids Research, Volume 36, Issue suppl_2, 1 July 2008, Pages W70-W74, DOI: 10.1093/nar/gkn188

Lorenz, R. and Bernhart, S.H. and Höner zu Siederdissen, C. and Tafer, H. and Flamm, C. and Stadler, P.F. and Hofacker, I.L. "ViennaRNA Package 2.0", Algorithms for Molecular Biology, 6:1 page(s): 26, 2011

Mathews DH, Disney MD, Childs JL, Schroeder SJ, Zuker M, Turner DH. (2004) Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc Natl Acad Sci U S A 101(19):7287-92.

O'Connell MR. Molecular Mechanisms of RNA Targeting by Cas13-containing Type VI CRISPR–Cas Systems. Journal of Molecular Biology. 2019;431(1):66-87. doi: https://doi.org/10.1016/j.jmb.2018.06.029.

Pearson LA, Hisbergues M, Börner T, Dittmann E, Neilan BA. Inactivation of an ABC transporter gene, mcyH, results in loss of microcystin production in the cyanobacterium Microcystis aeruginosa PCC 7806. Appl Environ Microbiol. 2004;70(11):6370-8. Epub 2004/11/06. doi: 10.1128/aem.70.11.6370-6378.2004. PubMed PMID: 15528494; PubMed Central PMCID: PMCPMC525210.