Like tagging your grey suitcase with a bright ribbon helps you spot it from among tens of others, tagging molecules with fluorescent probes gives certain functionalities to the molecules we try to study. We can now see where they reside in a cell, when they traverse, which other molecules they meet, what communication transpires between them, and so on.
For similar purposes and applying techniques in chemistry to biological molecules, Prof. S.G. Srivatsan’s group at IISER Pune develops probes to study nucleic acids.
In a recent paper, in collaboration with research groups of Dr. Souvik Maiti and Dr. Debojyoti Chakraborty from the Institute of Genomics and Integrative Biology (CSIR-IGIB), the group has developed a technology, named as sgRNA-Click, to display small molecules to specific gene targets. The team has used click chemistry and the CRISPR-Cas gene targeting and editing technology to achieve this.
A type of molecular scissors, the CRISPR-Cas9 system is originally from a bacterial defense mechanism against viruses and has been harnessed to edit genomes at specific sites. The gene targeting takes place in the presence of a complex made up of a protein known as dCas9 (a mutant form of Cas9 protein that does not cut nucleic acids, but can target and possibly alter gene function) and a single guide RNA (sgRNA). sgRNA contains the complementary part of a particular sequence of the target DNA, which when complexed with dCas9 gets localized on the target gene locus.
“While methods are developed for localizing proteins through widely used expression platforms, analogous techniques for site-specific display of small molecules are not adequate and complicated,” says Prof. S.G. Srivatsan on the motivation behind this work.
The sgRNA-Click technology developed by the group uses bioorthogonal azide−alkyne click chemistry. Using this labeling approach, they first modified the 3’ end of a sgRNA sequence with azide-based nucleotide analogues in the presence of an enzyme named terminal uridylyl transferase (TUTase). Subsequently, the specific reaction between azide groups on sgRNA and an alkyne counterpart containing the desired small molecule allowed the functionalization of the gene target.
“This technique is modular, as drug molecules, effector molecules, fluorescent probes can be attached to the alkyne part, which would expand the therapeutic and diagnostic potential of this CRISPR-based system,” says Prof. Srivatsan.
The approach is versatile, in that it can be employed on any sgRNA of choice. A challenge the team faced while developing this technique was to do with its implementation over highly structured sgRNA. To overcome this, Jerrin Thomas George, graduate student working with Prof. Srivatsan, remodeled the sequence of sgRNA under study in a way that the function of the molecule was not perturbed.
The group is working further on understanding the mechanism behind the incorporation of azide molecules on sgRNA through TUTase enzyme and their subsequent modification using various biophysical methods.
This research received funding from the DBT and from Wellcome Trust-DBT India Alliance.
Terminal Uridylyl Transferase mediated site-directed access to clickable chromatin employing CRISPR-dCas9. Jerrin Thomas George, Mohd. Azhar, Meghali Aich, Dipanjali Sinha, Uddhav B. Ambi, Souvik Maiti, Debojyoti Chakraborty, and Seergazhi G. Srivatsan, J. Am. Chem. Soc. (2020) 142(32): 13954–13965.
- Reported by Dr. Suchibrata Borah, Edited by Dr. Shanti Kalipatnapu