Papers by Debojyoti Chakraborty
Nucleic acid detection and variant calling through CRISPR-based diagnostics (CRISPRDx) has facili... more Nucleic acid detection and variant calling through CRISPR-based diagnostics (CRISPRDx) has facilitated clinical decision-making, particularly during the COVID-19 pandemic. This has been further accelerated through the discovery of newer and engineered CRISPR effectors, expanding the portfolio of such diagnostic applications to a wide variety of pathogenic and non-pathogenic conditions. However, each diagnostic CRISPR pipeline requires customized detection schemes originating from fundamental principles of the Cas protein used, its guide RNA (gRNA) design parameters, and the assay readout. This is particularly relevant for variant detection, an attractive low-cost alternative to sequencing-based approaches for which no in silico pipeline for the ready-to-use design of CRISPR-based diagnostics currently exists. In this manuscript, we fill this lacuna using a unified webserver CriSNPr (CRISPR based SNP recognition), which provides the user the opportunity to de-novo design gRNAs based ...
American Journal of Medical Genetics Part C: Seminars in Medical Genetics, 2020
Oligodendrocytes in the central nervous system (CNS) produce myelin sheaths that insulate axons t... more Oligodendrocytes in the central nervous system (CNS) produce myelin sheaths that insulate axons to facilitate efficient electrical conduction. These myelin sheaths contain lamellar microtubules that enable vesicular transport into the inner sheath. Mechanistically, oligodendrocytes rely on Golgi outpost organelles and the associated protein tubulin polymerization promoting protein (TPPP) to nucleate or form new microtubules outside of the cell body. Consequently, elongation of lamellar microtubules is defective in Tppp knockout (KO) mice, which have thinner and shorter myelin sheaths. We now explore the behavioral phenotypes of Tppp KO mice using a number of different assays. In open-field assays, Tppp KO mice display similar activity levels and movement patterns as wild-type mice, indicating that they do not display anxiety behavior. However, Tppp KO mice lack fear responses by two types of assays, traditional fear-conditioning assays and looming fear assays, which test for innate fear responses. Deficits in fear conditioning, which is a memory-dependent task, as well as in spatial memory tests, support possible short-term memory defects in Tppp KO mice. Together, our experiments indicate a connection between CNS myelination and behavioral deficits.
Clustered regularly interspaced short palindromic repeat/CRISPR-associated (CRISPR Cas) is a comp... more Clustered regularly interspaced short palindromic repeat/CRISPR-associated (CRISPR Cas) is a component of the prokaryotic adaptive immune system which has been repurposed for genome editing in recent years. The precision, simplicity and flexibility of this system have opened up a broad range of biological applications covering basic research to biotechnology and medicine. Additionally, the multiplexing capabilities of CRISPR offers a promising approach for modeling or correcting common polygenic disorders along with its monogenic and infectious counterparts. Although CRISPR is not completely precise and questions remain regarding its specificity and modes of delivery, the robustness and wide applicability of this genome editing tool has opened up numerous ways to address these issues. In this chapter, we will discuss about initial progress toward CRISPR therapeutics, existing delivery modalities, the challenges before CRISPR editing before it becomes a therapeutic possibility and th...
Trends in genetics : TIG, 2021
Next-generation sequencing (NGS) has identified disease hallmarks and catalogued a vast reservoir... more Next-generation sequencing (NGS) has identified disease hallmarks and catalogued a vast reservoir of genetic information from humans and other species. Precise nucleotide-interrogation properties of clustered regularly interspaced short palindromic repeats (CRISPR) proteins have been harnessed to rapidly identify DNA-RNA signatures for diverse applications, bypassing the cost and turnaround times associated with diagnostic NGS.
Nature Biotechnology, 2021
Life Science Alliance, 2020
The RNA polymerase II (RNAPII) associated factor 1 complex (Paf1C) plays critical roles in modula... more The RNA polymerase II (RNAPII) associated factor 1 complex (Paf1C) plays critical roles in modulating the release of paused RNAPII into productive elongation. However, regulation of Paf1C-mediated promoter-proximal pausing is complex and context dependent. In fact, in cancer cell lines, opposing models of Paf1Cs’ role in RNAPII pause-release control have been proposed. Here, we show that the Paf1C positively regulates enhancer activity in mouse embryonic stem cells. In particular, our analyses reveal extensive Paf1C occupancy and function at super enhancers. Importantly, Paf1C occupancy correlates with the strength of enhancer activity, improving the predictive power to classify enhancers in genomic sequences. Depletion of Paf1C attenuates the expression of genes regulated by targeted enhancers and affects RNAPII Ser2 phosphorylation at the binding sites, suggesting that Paf1C-mediated positive regulation of pluripotency enhancers is crucial to maintain mouse embryonic stem cell sel...
The clinical success of CRISPR therapies is dependent on the safety and efficacy of Cas proteins.... more The clinical success of CRISPR therapies is dependent on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) has negligible affinity for mismatched substrates enabling it to discriminate off-targets in DNA with very high precision even at the level of binding. However, its cellular targeting efficiency is low, limiting its use in therapeutic applications. Here, we rationally engineer the protein to develop enhanced (enFnCas9) variants and expand its cellular editing activity to genomic loci previously inaccessible. Notably, some of the variants release the protospacer adjacent motif (PAM) constraint from NGG to NGR/NRG making them rank just below SpCas9-RY and SpCas9-NG in their accessibility across human genomic sites. The enFnCas9 proteins, similar to Cas12a and Cas12f, harbor high intrinsic specificity and can diagnose single nucleotide variants accurately. Importantly, they provide superior outcomes in terms of editing efficiency, knock-in rates,...
ABSTRACTThe COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacte... more ABSTRACTThe COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacted global health, causing increased mortality among elderly patients and individuals with comorbid conditions. During the passage of the virus through affected populations, it has undergone mutations- some of which have recently been linked with increased viral load and prognostic complexities. Interestingly, several of these variants are point mutations that are difficult to diagnose using the gold standard quantitative real-time PCR (qPCR) method. This necessitates widespread sequencing which is expensive, has long turn-around times, and requires high viral load for calling mutations accurately. In this study, we show that the high specificity of Francisella novicida Cas9 (FnCas9) to point mismatches can be successfully adapted for the simultaneous detection of SARS-CoV2 infection as well as for detecting point mutations in the sequence of the virus obtained from patient samples. We rep...
eLife, 2021
The COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacted global... more The COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacted global health, causing increased mortality among elderly patients and individuals with comorbid conditions. During the passage of the virus through affected populations, it has undergone mutations, some of which have recently been linked with increased viral load and prognostic complexities. Several of these variants are point mutations that are difficult to diagnose using the gold standard quantitative real-time PCR (qRT-PCR) method and necessitates widespread sequencing which is expensive, has long turn-around times, and requires high viral load for calling mutations accurately. Here, we repurpose the high specificity of Francisella novicida Cas9 (FnCas9) to identify mismatches in the target for developing a lateral flow assay that can be successfully adapted for the simultaneous detection of SARS-CoV-2 infection as well as for detecting point mutations in the sequence of the virus obtained ...
Ophthalmic Genetics, 2021
The unsustainability of sustainability wants to underline the improper and widespread use of a co... more The unsustainability of sustainability wants to underline the improper and widespread use of a complex concept that cannot be trivialized through the proposal of technological solutions. Although technological solutions represent "comfortable" tools for social reassurance, the risk is a social drift generated by the phenomenon of the "granfalloon". Behind the technological solutions there are new market proposals, which have nothing to do with the real resolution of the sustainability of the global socioeconomic system. The perception of the whole socioeconomic system as a single metabolic socio-ecological system, makes us interpret the role of technological solutions differently. The knowledge we have gained over the last 50 years on the functioning of natural systems represent consolidated bases for interpreting and providing solutions on the sustainability of mankind. This perception, while providing uncomfortable solutions, should change the perception of applied ecology within the academy community. Therefore, applied ecology no longer as a study of human effects on the ecosphere, but as a discipline that teaches mankind how to structure its socioeconomic metabolism compatibly with the constraints imposed by the ecosphere.
The Lancet, 2021
Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on ... more Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre-including this research content-immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
ABSTRACTRapid detection of pathogenic sequences or variants in DNA and RNA through a point-of-car... more ABSTRACTRapid detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for accelerated clinical prognosis as has been witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are difficult to implement in settings with limited resources necessitating the development of accurate alternative testing strategies that perform robustly. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a direct Cas9 based enzymatic readout for detecting nucleotide sequences and identifying nucleobase identity without the requirement of trans-cleavage activity of reporter molecules. We demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs) including heterozygous carriers of a mutation and present a simple design strategy in the form of a web-tool, JATAYU, for its implementation. FELUDA is semi quantitative, can be adapted to multiple signal d...
Journal of Pharmaceutical Innovation, 2020
Most studies based on siRNA or CRISPR/Cas9 systems rely on viral vectors for their transfection. ... more Most studies based on siRNA or CRISPR/Cas9 systems rely on viral vectors for their transfection. However, these viral vectors are immunogenic, which limits their application in gene therapy. Thus, identification of novel vectors with higher safety and improved targeting is desirable. Hence, we have explored the potential of biodegradable, chitosan coated PLGA nanocarriers for intracellular delivery of CRISPR/Cas9, and siRNA. In this investigation, we have compared the efficiency of chitosan-coated PLGA NPs (CS-PLGA NPs) with that of the PLGA NPs for the delivery of CRISPR and siRNA. Presented here is the preparation and evaluation of specifically surface-modified CS-PLGA NPs and PLGA NPs on their efficacy for binding and delivery of siRNA and CRISPR/Cas9 complex. Cy3 siRNA loaded on PLGA NPs showed an internalization of 4.6% and mean fluorescent intensity (MFI) of 13.76%, while that of CS PLGA resulted in 89% internalization and MFI of 67.95%. The in vitro GFP silencing assessed by anti-GFP siRNA and NPs resulted in 10–15% silencing by PLGA NPs and 50–55% silencing by CS-PLGA NPs. The GFP silencing by CRISPR-Cas9 plasmid pX459 with CS PLGA was 80–83%, while that of PLGA was 11% and of commercial Lipofectamine agent was 13%. The biodegradable CS-PLGA NPs exhibited successful loading and high binding efficiency for siRNA as well as CRISPRCas9 and resulted in effective silencing. Our studies report that the CS-PLGA NPs can be a novel suitable candidate for the effective delivery of siRNA and CRISPR/Cas9 complex.
Locus-specific interrogation of the genome using programmable CRISPR-based technologies is tremen... more Locus-specific interrogation of the genome using programmable CRISPR-based technologies is tremendously useful in dissecting the molecular basis of target gene function and modulating its downstream output. Although these tools are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using this technique is not well developed due to inadequate labeling technologies. Here, we report the development of a modular technology, sgRNA-Click (sgR-CLK), which harnesses the power of bioorthogonal click chemistry for remodeling CRISPR to display synthetic molecules on target genes. A terminal uridylyl transferase (TUTase) was repurposed to construct an sgRNA containing multiple minimally invasive bioorthogonal clickable handles, which served as a Trojan horse on CRISPR-dCas9 system to guide synthetic tags site-specifically on chromatin employing copper-catalyzed or strain-promoted click reactions. Our results demonstrate that sgR-CLK could provide a...
Locus-specific interrogation of target genes employing functional probes such as proteins and sma... more Locus-specific interrogation of target genes employing functional probes such as proteins and small molecules is paramount in decoding the molecular basis of gene function and designing tools to modulate its downstream effects. In this context, CRISPR-based gene editing and targeting technologies have proved tremendously useful as they can be programmed to target any gene of interest by simply changing the sequence of the single guide RNA (sgRNA). Although these technologies are widely utilized in recruiting genetically encoded functional proteins, display of small molecules using CRISPR system is not well developed due to the lack of adequate techniques. Here, we have devised an innovative technology called sgRNA-Click (sgR-CLK) that harnesses the power of bioorthogonal click chemistry for remodeling guide RNA to display synthetic molecules on target genes. sgR-CLK employs a novel posttranscriptional chemo-enzymatic labeling platform wherein a terminal uridylyl transferase (TUTase) was repurposed to generate clickable sgRNA of choice by site-specific tailoring of multiple azide-modified nucleotide analogs at the 3' end. The presence of a minimally invasive azide handle assured that the sgRNAs are indeed functional. Notably, an azide-tailed sgRNA targeting the telomeric repeat served as a Trojan horse on CRISPR-dCas9 system to guide synthetic tags (biotin) site-specifically on chromatin employing copper-catalyzed or strain-promoted click reactions. Taken together, sgR-CLK presents a significant advancement on the utility of bioorthogonal chemistry, TUTase and CRISPR toolbox, which could offer a simplified solution for site-directed display of small molecule probes and diagnostic tools on target genes.
Detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic a... more Detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for rapid clinical prognosis. In recent times, CRISPR based detection of nucleic acids has provided an economical and quicker alternative to sequencing-based platforms which are often difficult to implement in the field. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a highly accurate enzymatic readout for detecting nucleotide sequences, identifying nucleobase identity and inferring zygosity with precision. We demonstrate that FELUDA output can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications including rapid diagnosis during infectious disease outbreaks like COVID-19.
Current Topics in Developmental Biology, 2019
Viscoelastic silicone rubber (VSR) is a remarkable shape-memory solid. The material's polymer net... more Viscoelastic silicone rubber (VSR) is a remarkable shape-memory solid. The material's polymer network retains a memory of its shape history, so its current and future shapes depend strikingly on its past shapes. Although VSR's memory fades gradually and it has a permanent (cured-in) shape to which it will eventually return when left alone, VSR can be taught new shapes and retain them for significant lengths of time. To examine VSR's ability to learn, remember, and recover shapes, this work focuses on a simple experiment. A VSR that has relaxed into its permanent shape is suddenly compressed to about 80% of its original height. After a specific period of compression, the VSR is released and allowed to return to its permanent shape. Having learned a new shape during the compression period, however, the VSR is reluctant to return and takes seconds, minutes, or hours to do so, depending on how long it was compressed. In addition to observing these behaviors experimentally in VSR, we show that those behaviors are well-described by a simple viscoelastic model. Unlike typical viscoelastic models, which are constructed from integer-order viscoelastic elements (e.g. elastic springs and viscous dashpots), the model describing VSR is the Fractional Zener model and involves a fractional-order element known as a spring-pot. Here "fractional" refers to the branch of mathematical analysis known as fractional calculus, a discipline that deals with derivatives, integrals, and differential equations of non-integer order. For example, between the first derivative and a second derivative, there are an infinite number of fractional derivatives. Though well-developed and important, fractional calculus is far less familiar than integer calculus, so this article is necessarily somewhat pedagogical. For integer-order viscoelastic models and the materials they describe, the future depends only on the present. For fractional-order models, the future depends also on the past. That memory of the past is intrinsic to fractional time derivatives: the fractional time derivative of any function f (t) depends not only on f (t ) at times t infinitesimally close to time t, but also on f (t ) at all times t where t < t. Both VSR and the Fractional Zener model that describes its behaviors are acutely aware of the past. The model's mathematical machinery make it possible to design VSR behaviors based on physical parameters, although some of the model's relationships are not yet known in closed form. VSR's existence as a practical material means that devices can be designed and produced that use a memory of past shapes to do things that would otherwise be difficult or impossible to make.
Genome editing using the CRISPR Cas9 system has been used to manipulate eukaryotic DNA and make p... more Genome editing using the CRISPR Cas9 system has been used to manipulate eukaryotic DNA and make precise heritable changes. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene- editing applications across different platforms, concerns remain, regarding their putative off targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when up to two mismatches to the target sgRNA are present in the sgRNA:DNA heteroduplex. In vivo, FnCas9 can be used for NHEJ mediated gene disruption and HDR mediated genomic integration. We propose that FnCas9 can be used for precise therapeutic genome editing.
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Papers by Debojyoti Chakraborty