Part:BBa_K3982041

CODE M Construct S5

This construct has been used in Project CODE M by Team IISER_Berhampur 2021. The aim of this project is to combat Multidrug-Resistant Tuberculosis (MDR-TB) with our diagnostic kit CODE M using mobile phone microscopy. Mutant strains of Mycobacterium tuberculosis, the causative agent of MDR-TB (which confer drug resistance towards two main drugs for TB cure - isoniazid and rifampicin) are detected through high-fidelity SNP detection using CRISPR/Cas technology. Here we used Cas14a1 (also called as Un1Cas12f1) as the RNA guided nuclease with our set of customised CODE M sgRNAs.

This is a standard construct has been assembled using various new basic parts as well as existing parts to synthesize the customized sgRNA in-vitro for use with Cas14a1 protein. The purpose of this construct is to test the expression of our CODE M sgRNAs with some common regulatory parts.

Usage and Biology

CRISPR/Cas technology has been derived from the CRISPR/Cas systems present in archaea and bacteria. They function as an adaptive immune system against invading foreign DNA and RNA. This technology has grown in the past several years with a wide range of applications. There are different types of Cas proteins such as Cas9, Cas12, Cas13 as well as Cas14. All of them are RNA guided nucleases - with a crRNA and tracrRNA or a sgRNA.

sgRNAs have significantly evolved from the natural guide RNAs which consisted of two parts - crRNA and tracrRNA. The optimized structure of sgRNA makes it highly efficient for genome editing comparable to that of spCas9 guide RNAs.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 406
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]