RNA

Part:BBa_K3602003:Design

Designed by: Flora Fuglsang   Group: iGEM20_SDU-Denmark   (2020-10-23)


rs6983267 T, sgRNA Cas13a


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The scaffold sequence was designed to match the requirements for cas13a according to Gootenberg et al. 2017. (https://science.sciencemag.org/content/356/6336/438)

The T7 promoter was chosen from neb.com for high yield RNA synthesis. (https://international.neb.com/protocols/2015/11/24/sgrna-synthesis-using-the-hiscribe-quick-t7-high-yield-rna-synthesis-kit-neb-e2050)

When designing the spacer sequence of our sgRNA, it was taken into consideration that optimal length is 28-31 nucleotides. Additionally, the rules regarding the tolerance on the seed sequence and mismatch for the Cas12a spacer sequence also applied to the Cas13a spacer sequence. Therefore, the only difference between the sgRNAs will be the scaffold sequence and the unspecific ribonuclease activity towards ssRNA instead of ssDNA.

Gootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017;356(6336):438-442. doi:10.1126/science.aam9321

Source

The sequence is designed from the ERG mRNA sequence from the human reference genome GRCh38 via ensembl (https://www.ensembl.org/).

References