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Part:BBa_K2306015

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


Cas13a spacer with flanking double repeats

This biobrick contains the sequence that encodes for the CRISPR array of Cas13a (Gootenberg et al. 2017). The CRISPR array encodes for the RNA guide used by the protein, making it able to find its target. This CRISPR array consists of a spacer flanked by two direct repeat sequences (DR). The spacer contains two BsaI restriction sites at each flank with 7 random bp between the two sites, which add up to 21 bp. The BsaI sites makes the user able to easily replace the target as one wishes.

Advised is to use a constitutive promoter for the transcription of the crRNA. We used Part:BBa_J23119 (see Part:BBa_K2306013 for the composite part). Also a terminator should be added downstream of the array.

When designing a new spacer to ligate into the array, make sure it contains the right sticky ends at both ends. One might consider to use our Cas13a crRNA design tool to design crRNAs that meet all the requirements.

Usage and Biology

This biobrick can be used in combination with parts encoding for Cas13a. From the this part CRISPR guide RNA (crRNA) will be synthesized. By restriction enzyme digestion with BsaI the spacer will be removed leaving two sticky ends. A new spacer can then be ligated in the array using sticky en ligation.

A promoter and terminator should be added. Sequence and Features

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


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

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