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Part:BBa_K2306015:Design

Designed by: Jeroen Jacques and Jasper Veerman   Group: iGEM17_TUDelft   (2017-10-30)


Cas13a spacer with flanking double repeats


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 51
    Illegal BsaI.rc site found at 38


Design Notes

We chose to use BsaI restriction site to create a spacer that can be cut out, because the cleavage site of BsaI lays upstream of the recognition site which make it possible to remove the entire spacer wihtout leaving traces. Furthermore BsaI creates sticky ends, preventing of self ligation of the CRISPR array.


Source

The spacer sequence consist of 21 bp. Each flank contains a BsaI restriction site so the spacer can easily be removed, leaving sticky ends where a new spacer can be inserted. Between the two BsaI restriction sites lays 7 random nucleotides, so that the entire length of the spacer would be 21 bp. The direct repeats sequence was taken from the plasmid pC011 from Gootenberg et al. 2017.

References

Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J., … Koonin, E. V. (2017). Nucleic acid detection with CRISPR-Cas13a/C2c2, 9321(April). https://doi.org/10.1126/science.aam9321