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.

Interaction of hnRNPB1 with Helix-12 of hHOTAIR Reveals the Distinctive Mode of RNA Recognition That Enables the Structural Rearrangement by LCD.

The lncRNA human Hox transcript antisense intergenic RNA (hHOTAIR) regulates gene expression by recruiting chromatin modifiers. The prevailing model suggests that hHOTAIR recruits hnRNPB1 to facilitate intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its target gene transcripts. This B1-mediated RNA-RNA interaction modulates the structure of hHOTAIR, attenuates its inhibitory effect on polycomb repression complex 2, and enhances its methyl transferase activity. However, the molecular details by which the nuclear hnRNPB1 protein assembles on the lncRNA HOTAIR have not yet been described. Here, we investigate the molecular interactions between hnRNPB1 and Helix-12 (hHOTAIR). We show that the low-complexity domain segment (LCD) of hnRNPB1 interacts with a strong affinity for Helix-12. Our studies revealed that unbound Helix-12 folds into a specific base-pairing pattern and contains an internal loop that, as determined by thermal melting and NMR studies, exhibits hydrogen bonding between strands and forms the recognition site for the LCD segment. In addition, mutation studies show that the secondary structure of Helix-12 makes an important contribution by acting as a landing pad for hnRNPB1. The secondary structure of Helix-12 is involved in specific interactions with different domains of hnRNPB1. Finally, we show that the LCD unwinds Helix-12 locally, indicating its importance in the hHOTAIR restructuring mechanism.

G4-binding drugs, chlorpromazine and prochlorperazine, repurposed against COVID-19 infection in hamsters.

The COVID-19 pandemic caused by SARS-CoV-2 has caused millions of infections and deaths worldwide. Limited treatment options and the threat from emerging variants underline the need for novel and widely accessible therapeutics. G-quadruplexes (G4s) are nucleic acid secondary structures known to affect many cellular processes including viral replication and transcription. We identified heretofore not reported G4s with remarkably low mutation frequency across >5 million SARS-CoV-2 genomes. The G4 structure was targeted using FDA-approved drugs that can bind G4s - Chlorpromazine (CPZ) and Prochlorperazine (PCZ). We found significant inhibition in lung pathology and lung viral load of SARS-CoV-2 challenged hamsters when treated with CPZ or PCZ that was comparable to the widely used antiviral drug Remdesivir. In support, in vitro G4 binding, inhibition of reverse transcription from RNA isolated from COVID-infected humans, and attenuated viral replication and infectivity in Vero cell cultures were clear in case of both CPZ and PCZ. Apart from the wide accessibility of CPZ/PCZ, targeting relatively invariant nucleic acid structures poses an attractive strategy against viruses like SARS-CoV-2, which spread fast and accumulate mutations quickly.

Biophysical Characterization of Nucleolin Domains Crucial for Interaction with Telomeric and TERRA G-Quadruplexes.

Nucleolin (NCL) is a well-characterized nucleic acid-binding protein; it binds to various canonical and noncanonical structures including single- and double-stranded DNA and RNA, hairpin, loops, and G-quadruplex structures. G-quadruplex structures are majorly formed in promoter, telomeric, and untranslated regions of the genome and affect the process of replication, transcription, and translation. One of the widely studied G-quadruplex-forming regions are telomeres, as these are sites for the recruitment for various proteins providing stability or having an effect on the telomerase activity. NCL is known to bind to both single- and double-stranded telomeric regions and its transcribed telomeric RNA (TERRA). In our study, we show that the 21nt G-quadruplex-forming region of telomeric DNA and TERRA RNA binds to NCL and the domains RRM1234 destabilize the telomeric G-quadruplex structure. We also show the preferential binding of the RNA G-quadruplex over the DNA G-quadruplex by two NCL domains, RRM3 and RRM4. Our findings provide insights into the binding preferences of RRM domains toward G-quadruplex structures and their subsequent effect on the quadruplex stability.

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/.

Small molecule quercetin binds MALAT1 triplex and modulates its cellular function.

The triple-helix structure at the 3' end of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA, has been considered to be a target for modulating the oncogenic functions of MALAT1. This study examines the binding of quercetin-a known triplex binding molecule-to the MALAT1 triplex. By employing UV-visible spectroscopy, circular dichroism spectroscopy, and isothermal titration calorimetry, we observed that quercetin binds to the MALAT1 triplex with a stoichiometry of 1:1 and K d of 495 ± 61 nM, along with a negative change in free energy, indicating a spontaneous interaction. Employing real-time PCR measurements, we observed around 50% downregulation of MALAT1 transcript levels in MCF7 cells, and fluorescence in situ hybridization (FISH) experiments showed concomitantly reduced levels of MALAT1 in nuclear speckles. This interaction is likely a result of a direct interaction between the molecule and the RNA, as indicated by a transcription-stop experiment. Further, transcriptome-wide analysis of alternative splicing changes induced by quercetin revealed modulation of MALAT1 downstream genes. Collectively, our study shows that quercetin strongly binds to the MALAT1 triplex and modulates its functions. It can thus be used as a scaffold for further development of therapeutics or as a chemical tool to understand MALAT1 functions.

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.

Fine-tuning miR-21 expression and inhibition of EMT in breast cancer cells using aromatic-neomycin derivatives.

MicroRNAs (miRs) are a class of endogenously expressed non-coding RNAs that negatively regulate gene expression within cells and participate in maintaining cellular homeostasis. By targeting 3' UTRs of target genes, individual miRs can control a wide array of gene expressions. Previous research has shed light upon the fact that aberrantly expressed miRs within cells can pertain to diseased conditions, such as cancer. Malignancies caused due to miRs are because of the high expression of onco-miRs or feeble expression of tumor-suppressing miRs. Studies have also shown miRs to engage in epithelial to mesenchymal transition (EMT), which allows cancer cells to become more invasive and metastasize. miR-21 is an onco-miR highly expressed in breast cancer cells and targets protein PTEN, which abrogates EMT. Therefore, we discuss an approach where in-house-developed peptidic amino sugar molecules have been used to target pre-miR-21 to inhibit miR-21 biogenesis, and hence antagonize its tumor-causing effect and inhibit EMT. Our study shows that small-molecule-based fine-tuning of miR expression can cause genotypic as well as phenotypic changes and also reinstates the potential and importance of nucleic acid therapeutics.

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.

Zinc2+ ion inhibits SARS-CoV-2 main protease and viral replication in vitro.

Zinc deficiency is linked to poor prognosis in COVID-19 patients while clinical trials with zinc demonstrate better clinical outcomes. The molecular targets and mechanistic details of the anti-coronaviral activity of zinc remain obscure. We show that zinc not only inhibits the SARS-CoV-2 main protease (Mpro) with nanomolar affinity, but also viral replication. We present the first crystal structure of the Mpro-Zn2+ complex at 1.9 Å and provide the structural basis of viral replication inhibition. We show that Zn2+ coordinates with the catalytic dyad at the enzyme active site along with two previously unknown water molecules in a tetrahedral geometry to form a stable inhibited Mpro-Zn2+ complex. Further, the natural ionophore quercetin increases the anti-viral potency of Zn2+. As the catalytic dyad is highly conserved across SARS-CoV, MERS-CoV and all variants of SARS-CoV-2, Zn2+ mediated inhibition of Mpro may have wider implications.

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.

Genome-Wide Computational Analysis and Validation of Potential Long Noncoding RNA-Mediated DNA-DNA-RNA Triplexes in the Human Genome.

Long noncoding RNAs are well studied for their regulatory actions through interaction with DNA regulating biological roles of DNA, RNA, or protein. However, direct binding of lncRNA with DNA is rarely demonstrated in experiments. The present protocol explains genome wide computational strategies to choose lncRNAs that can bind directly to the chromatin by forming highly stable DNA-DNA-RNA triplexes. The chapter also focuses on biophysical methods that can be used to validate the computationally derived lncRNA-gene targets in vitro.