Difference between revisions of "Part:BBa K3982008"

Latest revision as of 15:15, 21 October 2021

CODE M sgRNA for targeting mutated rpoB gene in Mycobacterium tuberculosis

This sequence encodes for the customised sgRNA and is used for targeting mutant rpoB gene in Mycobacterium tuberculosis.

Usage and Biology

Cas14a1 (also called as Un1Cas12f1) is a miniature cas protein used in CRISPR/Cas technology identified from archaebacteria and bacteriophages. It is a RNA guided nuclease which cleaves ssDNA without any Protospacer Adjacent Motif (PAM) specificity i.e. PAM independent ssDNA cleavage. This enables high-fidelity Single-nucleotide polymorphism (SNP) genotyping. It is guided by a single guide RNA (sgRNA) which has two components - CRISPR RNA (crRNA) and Trans-activating crispr RNA (tracrRNA).

crRNA is a 20bp nucleotide sequence complementary to the target DNA. tracrRNA is linked to the crRNA with a tetraloop that is responsible for cas14 activity. It plays a role in maturation of crRNA and has a stem-loop structure in itself that binds to the ribonuclease. Click here to know more about how this part is used in project CODE M by Team IISER_Berhampur 2021.

This sgRNA has been designed specifically for targeting the mutant rpoB gene along with Cas14a1 protein in Mycobacterium tuberculosis (Mtb) which confers to rifampicin drug resistance in Multidrug-resistant Tuberculosis (MDR-TB). The mutation appears at base number 11 (tcg → ttg) of the spacer (guiding) sequence. This is the S450L mutation in Mtb genome (refer Ref 3).

Refer to the wildtype rpoB targeting sgRNA here - BBa_K3982007

Sequence and Features