Difference between revisions of "Part:BBa K3799026"

Revision as of 01:47, 22 October 2021

Type III-A CRISPR-Cas effector ribonuclease---Csm6

Csm6 is a non-specific endoribonuclease that cleaves single-stranded RNA upon activation by cyclic adenylates or linear adenine homopolymers terminated with a 2'3'-cyclic phosphate.

Sequence and Features

Usage and Biology

Description - EiCsm6 is obtained from Enterococcus italicus, and belongs to the type III CRISPR systems. Csm6 is mainly activated by cyclic oligoadenylate second messengers by binding to their CARF domain. This activates the HEPN domain of the Csm6 for collateral RNA degradation. The CARF domain then degrades the cyclic adenylate activator (cA6) which functions as an off switch to regulate the activity of the CRISPR enzyme. These mechanisms facilitate rapid invader clearance and ensures proper termination to limit self toxicity and suicide of the bacteria.

EiCsm6 is coupled with the enzyme LwaCas13a(BBa_K2323004) to create the detector system SHERLOCKv2. The overhangs that are produced by the cleavage of RNA by Cas13a produces 2’-3’ cyclic phosphate hex adenylate residues that activate Csm6. This activation results in the collateral cleavage of RNA by both Cas13a and Csm6.

Cloning and characterization

Cloning:
From IDT, gene blocks with a 6x-His-tag, tobacco etch virus protease cut site, and restriction enzyme cut sites on the 5' and 3' end for NcoI and BlpI, respectively was oredered. After digesting the gene segments with restriction enzymes, they were ligated to the pET-52b expression vector. The assembled plasmid was transformed into a C41 pLysS cell line. Colonies were grown on selective media to screen the cells containing transformed plasmids containing Csm6 gene. Colony PCR was performed to make sure that the transformed plasmids were correct in insertion and orientation. [Image:T--IISER_Kolkata--image-Partnership-PCR.png | thumb | center | 800 px |Coloy PCR of EiCsm6 and TtCsm6]] T--IISER_Kolkata--image-Partnership-PCR2.png