Cas13a spacer with flanking double repeats (with constitutive promoter)
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI site found at 94
Illegal BsaI.rc site found at 81
When designing the spacer sequence, we made sure the guide RNA was compatible with Cas13a; the size (21 bp) and the fact that the spacer must not bind to the target where the target has a G base on the 3' end (PFS: protospacer flanking site).
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 double repeats sequence was taken from the plasmid pC011 from Gootenberg et al. 2017.
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