Recommendations for designing gRNA for MB-CasFire:
Target Site
5’ NNNYYNGGGGGGGGGGGGGGGGGGGGNNNN 3’
3’ NNNRRNCCCCCCCCCCCCCCCCCCCCNNNN 5’
gRNA:
5’ GAAUUUCUACUAUUGUAGAUGGGGGGGGGGGGGGGGGGGG
- Length of the gRNA is 40 nt (including the repeat sequence).
- The following repeat sequence is included in the gRNA 5’-GAAUUUCUACUAUUGUAGAU-3’
- Location of the gRNA is downstream of YYN PAM site.
- gRNA does not contain the PAM sequence.
LAMP Primer considerations:
- Optimal gRNAs fall between the F1c and B1c primers with no overlap with these sequences. If necessary, gRNAs can be designed with 1-4 nts overlap.
- gRNAs can be contained in the loop, if necessary, but be mindful to avoid overlap with loop primers.
- Sequence overlap on PAM side should be avoided.
- Extensive overlap of LAMP primers and gRNA in the same direction should be avoided.
- Overlap of complementary gRNA and primer sequences should be minimized.
