Bio Patrika interviews Dr. George on his thoughts about “the application of innovative technology sgRNA-Click combined with CRISPR-dCas9”

Dr. Jerrin Thomas George’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Dr. George completed integrated MS-PhD from the IISER Pune. He pursued his doctoral research in the group of Prof. Seergazhi G. Srivastan on developing chemo-enzymatic platforms to functionalize RNA with biophysical probes using template dependent and independent polymerases. He then had a short stay as a research associate at JNCASR, Bangalore in the group of Dr. Sarit S. Agasti and now plans to join Columbia University, New York as a postdoctoral research scientist. His research interest is in the broad area of chemical biology, and molecular biophysics. For his future carrier, he is interested in deciphering the molecular mechanism of novel gene editing tools using various biophysical techniques. Jerrin published part of his PhD work titled “Terminal uridylyl transferase mediated site-directed access to clickable chromatin employing CRISPR-dCas9” as first author in Journal of the American Chemical Society (2020).

How would you explain your paper’s key results to the non-scientific community?

Interrogation of a target gene locus with various molecular tags and biophysical probes has a tremendous impact in gaining fundamental insight into a gene’s biological functions. Traditionally, this is achieved using various systems such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), which require extensive protein engineering to facilitate each gene of interest manipulation. On the other hand, the latest gene editing/targeting tool, clustered regularly interspaced short palindromic repeats (CRISPR)−CRISPR-associated proteins (Cas) simplifies the existing strategies for target gene manipulation by simply varying the sequence of a guide RNA. This enables the recruitment of various functional proteins directly on the intended gene. To achieve this, a mutant DNA targeting Cas protein, dCas9 is tagged with functional proteins such as epigenetic modulators, transcription activators, pull-down probes, and fluorescent probes. Although recruitment of a functional protein on a specific gene utilizing CRISPR-dCas9 technology is fairly well established, the attachment of synthetic tags onto a specific protein demands new strategies that allow the direct covalent conjugation of small molecules on gene targeting system. As a prerequisite, the conjugation of a covalent tag should not functionally inactivate the CRISPR-dCas9 system.

In our paper, we have developed an innovative technology namely, sgRNA-Click (sgR-CLK) to display synthetic molecules on a target gene of interest combining CRISPR-dCas9, bioorthogonal click chemistry and a posttranscriptional RNA modifying enzyme namely, terminal uridylyl transferase (TUTase). We tailor the single guide RNA (sgRNA) of the gene targeting tool with multiple click tags (azide in this case) using the TUTase and a modified nucleotide, 5-azidomethyl uridine triphosphate (AMUTP) (Figure 1). Further, we utilize the azides as handles for recruiting small molecules. The minimally invasive nature of the azide tags on the sgRNA preserve the activity of CRISPR-dCas9 for gene targeting and enable the multimeric recruitment of synthetic molecules on the target gene utilizing bioorthogonal click chemistries such as copper-catalyzed and strain-promoted azide-alkyne cycloaddition reactions. Markedly, our technique, sgR-CLK opens up new avenues for site-specific display of molecular probes and diagnostic tools onto a target gene.

Figure 1. The CRISPR sgRNA was terminal uridylated with AMUTP utilizing TUTase enzyme. The azide-tailed sgRNA is recruited to gene of interest by dCas9. The localization of azide-tagged sgRNA to the target gene is visualized in cells as intense nuclear punctate dots (green).

What are the possible consequences of these findings for your research area?

Gene targeting utilizing CRISPR-dCas9 has been used extensively in recent years for recruiting functional proteins on the target gene of interest achieved by genetically encoding dCas9 fused with the intended protein. However, strategies for recruiting synthetic molecules on a gene locus using CRISPR-dCas9 require direct covalent tagging of either the dCas9 protein or its sgRNA, which is technically cumbersome due to the lack of adequate labelling technologies. sgR-CLK repurposes a posttranscriptional RNA labelling enzyme, TUTase for covalently tailoring sgRNA with azide tags while preserving the overall activity of the gene targeting tool. Importantly, this technique provides a significant advancement for the locus-specific recruitment of synthetic drugs, photoactivatable groups, pull-down probes, epigenome and transcription modulators. Access to synthetic molecular probes and diagnostic tools on a target gene of interest will profoundly help gain fundamental understanding into various gene functions and modulate its downstream effects, important in biomedical research.

“[…] this technique provides a significant advancement for the locus-specific recruitment of synthetic drugs, photoactivatable groups, pull-down probes, epigenome and transcription modulators.”

What was the exciting moment (eureka moment) during your research?

In our pursuit for developing a technique to recruit synthetic molecules using CRISPR-dCas9, we circumvent many obstacles which we had to troubleshoot. An important obstacle was when we observed that click modification of sgRNA prior to assembly of the ternary complex, inhibited CRISPR-dCas9 activity. Upon further investigation in vitro, we observed that azide-tailored sgRNAs were functional, whereas click-functionalized sgRNA exhibited limited activity. Our eureka moment was when we visualized intense nuclear punctate dots, while performing confocal microscopy, indicating the localization of azide-tagged sgRNA to target genes in cells almost similar to the unmodified sgRNA. This gave us the confidence to further perform bioorthogonal click chemistry on the CRISPR ternary complex for displaying synthetic molecules on gene targets.

Where do you seek scientific inspiration?

The field of gene editing/targeting has seen a revolutionary change with the inception of bacterial CRISPR-Cas based technologies. These technologies have tremendously assisted in DNA base-editing, long-term gene tracking and in developing diagnostic platforms. Astounding research works from various groups worldwide on advancing human health utilizing gene editing platforms serve every day as a scientific inspiration to actively pursue new ideas and implement them. Indeed, hailing from a family with an academic background and pursuing my PhD with Prof. Srivatsan, an enthusiastic mentor, always gives me scientific inspiration.

What do you hope to do next?

Our lab will be further expanding sgR-CLK for live-cell gene targeting and applying this technology in targeting a specific cell type. An interesting feature of sgR-CLK is the ability to multiplex utilizing orthogonal click chemistries (with multiple reactive handles) and interrogate several genes in parallel that we are curious to explore. In our article in JACS, we displayed synthetic tags on a repetitive sequence. Next, our lab aims to expand sgR-CLK on non-repetitive targets and in recruit large synthetic cargos such as oligomers to target genes.

How do you intend to help Indian science improve?

In my opinion, a researcher of my age group can contribute to the progress of Indian science by generating novel ideas and translating them into high quality science, which will ultimately help mankind. I must admit that the progress made in my research is a collective effort of my colleagues at Prof. Srivatsan’s lab at IISER Pune and our collaborators at CSIR-IGIB, New Delhi (Dr. Souvik Maiti’s and Dr. Debojyoti Chakraborty’s lab). We are one of the few labs in India working on developing innovative technologies based on CRISPR-Cas systems. In addition to developing technologies such as sgR-CLK, our lab develops new tools for gaining a fundamental molecular understanding of nucleic acid structures in the cell. We, as a lab, help in advancing Indian science by developing new techniques and therapeutic platforms.

Reference

George, J. T.; Azhar, M.; Aich, M.; Sinha, D.; Ambi, U. B.; Maiti, S.; Chakraborty, D.; Srivatsan, S. G. Terminal uridylyl transferase mediated site-directed access to clickable chromatin employing CRISPR-dCas9. J. Am. Chem. Soc. 2020, 142, 13954–13965.

Google scholar link: https://scholar.google.com/citations?user=iDFc4psAAAAJ&hl=en

Email: jerrin.t.george@gmail.com

Learn more about lab of Prof. Srivatsan lab: https://www.iiserpune.ac.in/~srivatsan/

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