Rare genetic disorders in India: Current status, challenges, and CRISPR-based therapy.

Rare genetic diseases are a group of life-threatening disorders affecting significant populations worldwide and posing substantial challenges to healthcare systems globally. India, with its vast population, is also no exception. The country harbors millions of individuals affected by these fatal disorders, which often result from mutations in a single gene. The emergence of CRISPR-Cas9 technology, however, has ushered in a new era of hope in genetic therapies. CRISPR-based treatments hold the potential to precisely edit and correct diseasecausing mutations, offering tailored solutions for rare genetic diseases in India. This review explores the landscape of rare genetic diseases in India along with national policies and major challenges, and examines the implications of CRISPR-based therapies for potential cure. It delves into the potential of this technology in providing personalized and effective treatments. However, alongside these promising prospects, some ethical considerations, regulatory challenges, and concerns about the accessibility of CRISPR therapies are also discussed since addressing these issues is crucial for harnessing the full power of CRISPR in tackling rare genetic diseases in India. By taking a multidisciplinary approach that combines scientific advancements, ethical principles, and regulatory frameworks, these complexities can be reconciled, paving the way for innovative and impactful healthcare solutions for rare diseases in India.

TOBF1 modulates mouse embryonic stem cell fate through regulating alternative splicing of pluripotency genes.

Embryonic stem cells (ESCs) can undergo lineage-specific differentiation, giving rise to different cell types that constitute an organism. Although roles of transcription factors and chromatin modifiers in these cells have been described, how the alternative splicing (AS) machinery regulates their expression has not been sufficiently explored. Here, we show that the long non-coding RNA (lncRNA)-associated protein TOBF1 modulates the AS of transcripts necessary for maintaining stem cell identity in mouse ESCs. Among the genes affected is serine/arginine splicing factor 1 (SRSF1), whose AS leads to global changes in splicing and expression of a large number of downstream genes involved in the maintenance of ESC pluripotency. By overlaying information derived from TOBF1 chromatin occupancy, the distribution of its pluripotency-associated OCT-SOX binding motifs, and transcripts undergoing differential expression and AS upon its knockout, we describe local nuclear territories where these distinct events converge. Collectively, these contribute to the maintenance of mouse ESC identity.

Identification of G-quadruplex structures in MALAT1 lncRNA that interact with nucleolin and nucleophosmin.

Nuclear-retained long non-coding RNAs (lncRNAs) including MALAT1 have emerged as critical regulators of many molecular processes including transcription, alternative splicing and chromatin organization. Here, we report the presence of three conserved and thermodynamically stable RNA G-quadruplexes (rG4s) located in the 3' region of MALAT1. Using rG4 domain-specific RNA pull-down followed by mass spectrometry and RNA immunoprecipitation, we demonstrated that the MALAT1 rG4 structures are specifically bound by two nucleolar proteins, Nucleolin (NCL) and Nucleophosmin (NPM). Using imaging, we found that the MALAT1 rG4s facilitate the localization of both NCL and NPM to nuclear speckles, and specific G-to-A mutations that disrupt the rG4 structures compromised the localization of both NCL and NPM in speckles. In vitro biophysical studies established that a truncated version of NCL (ΔNCL) binds tightly to all three rG4s. Overall, our study revealed new rG4s within MALAT1, established that they are specifically recognized by NCL and NPM, and showed that disrupting the rG4s abolished localization of these proteins to nuclear speckles.

CriSNPr, a single interface for the curated and de novo design of gRNAs for CRISPR diagnostics using diverse Cas systems.

CRISPR-based diagnostics (CRISPRDx) have improved clinical decision-making, especially during the COVID-19 pandemic, by detecting nucleic acids and identifying variants. This has been accelerated by the discovery of new and engineered CRISPR effectors, which have expanded the portfolio of diagnostic applications to include a broad range of pathogenic and non-pathogenic conditions. However, each diagnostic CRISPR pipeline necessitates customized detection schemes based on the fundamental principles of the Cas protein used, its guide RNA (gRNA) design parameters, and the assay readout. This is especially relevant for variant detection, a low-cost alternative to sequencing-based approaches for which no in silico pipeline for the ready-to-use design of CRISPRDx currently exists. In this manuscript, we fill this lacuna using a unified web server, CriSNPr (CRISPR-based SNP recognition), which provides the user with the opportunity to de novo design gRNAs based on six CRISPRDx proteins of choice (Fn/enFnCas9, LwCas13a, LbCas12a, AaCas12b, and Cas14a) and query for ready-to-use oligonucleotide sequences for validation on relevant samples. Furthermore, we provide a database of curated pre-designed gRNAs as well as target/off-target for all human and SARS-CoV-2 variants reported thus far. CriSNPr has been validated on multiple Cas proteins, demonstrating its broad and immediate applicability across multiple detection platforms. CriSNPr can be found at http://crisnpr.igib.res.in/.

Capturing nucleic acid variants with precision using CRISPR diagnostics.

CRISPR/Cas systems have the ability to precisely target nucleotide sequences and enable their rapid identification and modification. While nucleotide modification has enabled the therapeutic correction of diseases, the process of identifying the target DNA or RNA has greatly expanded the field of molecular diagnostics in recent times. CRISPR-based DNA/RNA detection through programmable nucleic acid binding or cleavage has been demonstrated for a large number of pathogenic and non-pathogenic targets. Combining CRISPR detection with nucleic acid amplification and a terminal signal readout step allowed the development of numerous rapid and robust nucleic acid platforms. Wherever the Cas effector can faithfully distinguish nucleobase variants in the target, the platform can also be extended for sequencing-free rapid variant detection. Some initial PAM disruption-based SNV detection reports were limited to finding or integrating mutated/mismatched nucleotides within the PAM sequences. In this review, we try to summarize the developments made in CRISPR diagnostics (CRISPRDx) to date emphasizing CRISPR-based SNV detection. We also discuss the applications where such diagnostic modalities can be put to use, covering various fields of clinical research, SNV screens, disease genotyping, primary surveillance during microbial infections, agriculture, food safety, and industrial biotechnology. The ease of rapid design and implementation of such multiplexable assays can potentially expand the applications of CRISPRDx in the domain of affinity-based target sequencing, with immense possibilities for low-cost, quick, and widespread usage. In the end, in combination with proximity assays and a suicidal gene approach, CRISPR-based in vivo SNV detection and cancer cell targeting can be formulated as personalized gene therapy.

FnCas9 Editor Linked Uniform Detection Assay for COVID-19.

The recent COVID-19 outbreak and pandemic of 2020 and its surveillance were implemented by quickly adapting the existing diagnostic methods to detect the SARS-CoV-2 RNA. While traditional methods for detecting pathogenic DNA and RNA have relied heavily on gold standard quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and sequencing-based methods, their shortcomings under resource-limited settings have emphasized the need of developing point-of-care (POC) diagnostics. Clustered regularly interspaced short palindromic repeats (CRISPR)-based detection systems provide a rapid and accurate alternative. Here, we describe a CRISPR-Cas9-based detection system FnCas9 Editor Linked Uniform Detection Assay (FELUDA) using a lateral flow test that can detect nucleobase and nucleotide sequences depending upon the stoichiometric-based binding of FnCas9 ribonucleoprotein complex (RNP)-target sequences. The assay has been optimized to be conducted within 1 h and shows 100% sensitivity and 97% specificity in clinical samples across a range of viral loads. The lateral strip results are read using the True Outcome Predicted via Strip Evaluation (TOPSE) smartphone application. This assay is versatile and can be optimized and adjusted to target various diseases.

Alternative splicing modulation mediated by G-quadruplex structures in MALAT1 lncRNA.

MALAT1, an abundant lncRNA specifically localized to nuclear speckles, regulates alternative-splicing (AS). The molecular basis of its role in AS remains poorly understood. Here, we report three conserved, thermodynamically stable, parallel RNA-G-quadruplexes (rG4s) present in the 3' region of MALAT1 which regulates this function. Using rG4 domain-specific RNA-pull-down followed by mass-spectrometry, RNA-immuno-precipitation, and imaging, we demonstrate the rG4 dependent localization of Nucleolin (NCL) and Nucleophosmin (NPM) to nuclear speckles. Specific G-to-A mutations that abolish rG4 structures, result in the localization loss of both the proteins from speckles. Functionally, disruption of rG4 in MALAT1 phenocopies NCL knockdown resulting in altered pre-mRNA splicing of endogenous genes. These results reveal a central role of rG4s within the 3' region of MALAT1 orchestrating AS.

Accelerating Adaptation of Forest Trees to Climate Change Using Individual Tree Response Functions.

In forest tree breeding, assisted migration has been proposed to accelerate the adaptive response to climate change. Response functions are currently fitted across multiple populations and environments, enabling selections of the most appropriate seed sources for a specific reforestation site. So far, the approach has been limited to capturing adaptive variation among populations, neglecting tree-to-tree variation residing within a population. Here, we combined the response function methodology with the in-situ breeding approach, utilizing progeny trials of European larch (Larix decidua) across 21 test sites in Austria ranging from Alpine to lowland regions. We quantified intra-population genetic variance and predicted individual genetic performance along a climatic gradient. This approach can be adopted in most breeding and conservation programs, boosting the speed of adaptation under climate change.

FnCas9-based CRISPR diagnostic for rapid and accurate detection of major SARS-CoV-2 variants on a paper strip.

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 from patient samples. We report the detection of the S gene mutation N501Y (present across multiple variant lineages of SARS-CoV-2) within an hour using lateral flow paper strip chemistry. The results were corroborated using deep sequencing on multiple wild-type (n = 37) and mutant (n = 22) virus infected patient samples with a sensitivity of 87% and specificity of 97%. The design principle can be rapidly adapted for other mutations (as shown also for E484K and T716I) highlighting the advantages of quick optimization and roll-out of CRISPR diagnostics (CRISPRDx) for disease surveillance even beyond COVID-19. This study was funded by Council for Scientific and Industrial Research, India.

SARS-CoV-2, the virus responsible for COVID-19, has a genome made of RNA (a nucleic acid similar to DNA) that can mutate, potentially making the disease more transmissible, and more lethal. Most countries have monitored the rise of mutated strains using a technique called next generation sequencing (NGS), which is time-consuming, expensive and requires skilled personnel. Sometimes the mutations to the virus are so small that they can only be detected using NGS. Finding cheaper, simpler and faster SARS-CoV-2 tests that can reliably detect mutated forms of the virus is crucial for public health authorities to monitor and manage the spread of the virus. Lateral flow tests (the same technology used in many pregnancy tests) are typically cheap, fast and simple to use. Typically, lateral flow assay strips have a band of immobilised antibodies that bind to a specific protein (or antigen). If a sample contains antigen molecules, these will bind to the immobilised antibodies, causing a chemical reaction that changes the colour of the strip and giving a positive result. However, lateral flow tests that use antibodies cannot easily detect nucleic acids, such as DNA or RNA, let alone mutations in them. To overcome this limitation, lateral flow assays can be used to detect a protein called Cas9, which, in turn, is able to bind to nucleic acids with specific sequences. Small changes in the target sequence change how well Cas9 binds to it, meaning that, in theory, this approach could be used to detect small mutations in the SARS-CoV-2 virus. Kumar et al. made a lateral flow test that could detect a Cas9 protein that binds to a nucleic acid sequence found in a specific mutant strain of SARS-CoV-2. This Cas9 was highly sensitive to changes in its target sequence, so a small mutation in the target nucleic acid led to the protein binding less strongly, and the signal from the lateral flow test being lost. This meant that the lateral flow test designed by Kumar et al. could detect mutations in the SARS-CoV-2 virus at a fraction of the price of NGS approaches if used only for diagnosis. The lateral flow test was capable of detecting mutant viruses in patient samples too, generating a colour signal within an hour of a positive sample being run through the assay. The test developed by Kumar et al. could offer public health authorities a quick and cheap method to monitor the spread of mutant SARS-CoV-2 strains; as well as a way to determine vaccine efficacy against new strains.

Low-cost CRISPR diagnostics for resource-limited settings.

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.

CRISPR Cas9 based genome editing in inherited retinal dystrophies.

BACKGROUND: Precision genome engineering, with targeted therapy towards patient-specific mutations is predicted to be the future of personalized medicine. Ophthalmology is in the frontiers of development of targeted therapy since the eye is an accessible organ and has the ease of both delivery as well as monitoring effects of therapy. MATERIALS AND METHODS: We reviewed literature using keywords CRISPR, precision medicine, genomic editing, retinal dystrophies, retinitis pigmentosa, Usher syndrome, Stargardt's Disease. Further, we collated data on current clinical trials. RESULTS: There is growing evidence on the role of genomic editing in retinal dystrophies, the various methods used, and stage of development of different therapies have been summarized in this paper. CONCLUSIONS: The CRISPR-Cas9 system has revolutionized genome editing, and opened avenues in drug discovery. It is important to understand the role of this system along with its applicability in the field of ophthalmology. In this review article, we briefly describe its methodology, the strategies of employing it for making genetic perturbations, and explore its applications in inherited retinal dystrophies.

Rapid and accurate nucleobase detection using FnCas9 and its application in COVID-19 diagnosis.

Rapid detection of DNA/RNA pathogenic sequences or variants through point-of-care diagnostics is valuable for accelerated clinical prognosis, as witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are tough to implement with limited resources, necessitating the development of accurate and robust alternative strategies. Here, we report FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a direct Cas9 based enzymatic readout for detecting nucleobase and nucleotide sequences without trans-cleavage of reporter molecules. We also demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs), including heterozygous carriers, and present a simple web-tool JATAYU to aid end-users. FELUDA is semi-quantitative, can adapt to multiple signal detection platforms, and deploy for versatile applications such as molecular diagnosis during infectious disease outbreaks like COVID-19. Employing a lateral flow readout, FELUDA shows 100% sensitivity and 97% specificity across all ranges of viral loads in clinical samples within 1hr. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection closer to home.

The Paf1 complex positively regulates enhancer activity in mouse embryonic stem cells.

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 self-renewal.

Ophthalmic genetics practice and research in India: Vision in 2020.

Ophthalmic genetics is a much needed and growing area in India. Ethnic diversity, with a high degree of consanguinity, has led to a high prevalence of genetic disorders in the country. As the second most populous country in the world, this naturally results in a significant number of affected people overall. Practice involves coherent association between ophthalmologists, genetic counselor and pediatricians. Eye genetics in India in recent times has witnessed advanced research using cutting edge diagnostics, next generation sequencing (NGS) approaches, stem cell therapies, gene therapy and genomic editing. This article will highlight the studies reporting genetic variations in the country, challenges in practice, and the latest advances in ophthalmic genetic research in India.

Terminal Uridylyl Transferase Mediated Site-Directed Access to Clickable Chromatin Employing CRISPR-dCas9.

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 chemoenzymatic 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 analogues 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 the 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 the CRISPR toolbox, which could offer a simplified solution for site-directed display of small molecule probes and diagnostic tools on target genes.

Role of lncRNAs in stem cell maintenance and differentiation.

Embryonic Stem cells are widely studied to elucidate the disease and developmental processes because of their capability to differentiate into cells of any lineage, Pervasive transcription is a distinct feature of all multicellular organisms and genomic elements such as enhancers and bidirectional or unidirectional promoters regulate these processes. Thousands of loci in each species produce a class of transcripts called noncoding RNAs (ncRNAs), that are well known for their influential regulatory roles in multiple biological processes including stem cell pluripotency and differentiation. The number of lncRNA species increases in more complex organisms highlighting the importance of RNA-based control in the evolution of multicellular organisms. Over the past decade, numerous studies have shed light on lncRNA biogenesis and functional significance in the cell and the organism. In this review, we focus primarily on lncRNAs affecting the stem cell state and developmental pathways.