Part:BBa_K3982005:Design

CODE M sgRNA for targeting mutant katG gene in Mycobacterium tuberculosis

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 region has been designed as referred from Ref. 1. It consists of the tracrRNA and some regions of crRNA. The guiding region is the 20 bp spacer sequence which is complementary to the mutant katG gene in Mtb and referred from Ref 2. The mutation appears at base number 11 (agc → acc) of the spacer (guiding) sequence. This is the S315T mutation in Mtb genome (refer Ref 3).

Refer to the wildtype katG targeting sgRNA here - BBa_K3982004

The 20 bp spacer sequence is as follows: gcgatcaccaccggcatcga

References

1) Kim, D.Y., Lee, J.M., Moon, S.B. et al. Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus. Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-01009-z

2) NCBI Reference Sequence for Mycobacterium tuberculosis H37Rv: NC_000962.3

3) Musser, J. M., Kapur, V., Williams, D. L., Kreiswirth, B. N., van Soolingen, D., & van Embden, J. D. (1996). Characterization of the catalase-peroxidase gene (katG) and inhA locus in isoniazid-resistant and -susceptible strains of Mycobacterium tuberculosis by automated DNA sequencing: restricted array of mutations associated with drug resistance. The Journal of infectious diseases, 173(1), 196–202. https://doi.org/10.1093/infdis/173.1.196