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.

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.

Microtubule alterations may destabilize photoreceptor integrity: Age-related microtubule changes and pattern of expression of MAP-2, Tau and hyperphosphorylated Tau in aging human photoreceptor cells.

Photoreceptor cells undergo changes with aging. It is unknown if their microtubules are stable or not with aging. This study examined photoreceptor cell ultrastructure from 18 human donor retinas (32 eyes; age: 45-94 years) and quantified the photoreceptors with altered microtubules over six to ninth decades in four defined retinal regions. In addition, immunoreactivity (IR) to microtubule-associated protein-2 (MAP-2), tau and hyperphophorylated tau was performed in retinal sections from companion eyes. In young donor retinas below 75 years of age, microtubules appeared straight in photoreceptor inner segments and axons. With age, they appeared bent or misaligned in macular and mid-peripheral photoreceptors. In addition, dense granular materials were present in photoreceptor axons and synaptic terminals in advanced ages. In all decades, rod microtubules were affected more than their cone counterparts (28% vs 15%, p < 0.005). Both rods and cones were significantly affected in mid-peripheral retina (5-8 mm outside the macular border) in eighth decade, compared to other decades or retinal regions (parafoveal, perifoveal and nasal) examined (p < 0.005). IR showed a steady expression of MAP-2 in inner segments, and tau in inner segments to axons below 75 years of age, but was absent for both markers in scattered macular and mid-peripheral photoreceptors in advanced ages (>75 years). IR to hyperphosphorylated tau was present mainly in inner retina and increased with aging. Markers of oxidative stress, e.g., lipid peroxidation (4-hydroxy 2-nonenal) and nitrosative stress (nitrotyrosine) were immunopositive in aged photoreceptors. The sporadic loss of MAP-2 and tau-IR in photoreceptors may be due to microtubule changes; all these changes may affect intracellular transport and be partly responsible for photoreceptor death in aged human retina.

Changes in the Inner Retinal Cells after Intense and Constant Light Exposure in Sprague-Dawley Rats.

Light insult causes photoreceptor death. Few studies reported that continuous exposure to light affects horizontal, Müller and ganglion cells. We aimed to see the effect of constant light exposure on bipolar and amacrine cells. Adult Sprague-Dawley rats were exposed to 300 or 3000 lux for 7 days in 12-h light: 12-h dark cycles (12L:12D). The latter group was then exposed to 24L:0D for 48 h to induce significant damage. The same animals were reverted to 300 lux and reared for 15 days in 12L:12D cycles. They were sacrificed on different days to find the degree of retinal recovery, if any, from light injury. Besides photoreceptor death, continuous light for 48 h resulted in downregulation of parvalbumin in amacrine cells and recoverin in cone bipolar cells (CBC). Rod bipolar cells (RBC) maintained an unaltered pattern of PKC-α expression. Upon reversal, there were increased expressions of parvalbumin in amacrine cells and recoverin in CBC, while RBC showed an increasing trend of PKC-α expression. The data show that damage in bipolar and amacrine cells after exposure to intense, continuous light can be ameliorated upon reversal to normal LD cycles to which the animals were initially acclimated to.

In ovo Sound Stimulation Mediated Regulation of BDNF in the Auditory Cortex and Hippocampus of Neonatal Chicks.

Brain-derived neurotrophic factor (BDNF) is known to mediate activity-dependent changes in the developing auditory system. Its expression in the brainstem auditory nuclei, auditory cortex and hippocampus of neonatal chicks (Gallus gallus domesticus) in response to in ovo high intensity sound exposure at 110 dB (arrhythmic sound: recorded traffic noise, 30-3000 Hz with peak at 2700 Hz, rhythmic sound: sitar music, 100-4000 Hz) was examined to understand the previously reported altered volume and neuronal number in these regions. In the brainstem auditory nuclei, no mature BDNF, but proBDNF at the protein level was detected, and no change in its levels was observed after in ovo sound stimulation (music and noise). Increased ProBDNF protein levels were found in the auditory cortex in response to arrhythmic sound, along with decreased levels of one of the BDNF mRNA transcripts, in response to both rhythmic and arrhythmic sound stimulation. In the hippocampus, increased levels of mature BDNF were found in response to music. Expression microarray analysis was performed to understand changes in gene expression in the hippocampus in response to music and noise, followed by gene ontology analysis showing enrichment of probable signaling pathways. Differentially expressed genes like CAMK1 and STAT1 were found to be involved in downstream signaling on comparing music versus noise-exposed chicks. In conclusion, we report that BDNF is differentially regulated in the auditory cortex at the transcriptional and post-translational level, and in the hippocampus at the post-translational level in response to in ovo sound stimulation.

Localization of nitro-tyrosine immunoreactivity in human retina.

Oxidative stress (OS) is associated with retinal aging and age-related macular degeneration (AMD). In both cases there are reports for the presence of markers of lipid peroxidation in retinal cells. We investigated if nitrosative stress also occurs in the human retina with aging. We examined the cellular localization of nitro-tyrosine, a biomarker of protein tyrosine nitration, in human donor retina (17-91 years; N = 15) by immunohistochemistry. Immunoreactivity (IR) to nitro-tyrosine was present in ten retinas and absent in five retinas. It was predominant in photoreceptor inner segments, cell bodies and axons. In six retinas, IR was present in abnormal, swollen axons of macular and peripheral cones. In the inner retina, weak immunoreactivity was detected in the outer and inner plexiform layer. Transmission electron microscopy revealed a variable degree of microtubule disorganization, abnormal outgrowth from the swollen macular axons (as the fibers of Henle) and few dead axons. The present study adds further evidence to the presence of aberrant photoreceptor axonal changes in the human retina and that nitro-tyrosine immunoreactivity is associated with the photoreceptor cells in select human retina.

Experimental oral iron administration: Histological investigations and expressions of iron handling proteins in rat retina with aging.

Iron is implicated in age-related macular degeneration (AMD). The aim of this study was to see if long-term, experimental iron administration with aging modifies retinal and choroidal structures and expressions of iron handling proteins, to understand some aspects of iron homeostasis. Male Wistar rats were fed with ferrous sulphate heptahydrate (500mg/kg body weight/week, oral; elemental iron availability: 20%) from 2 months of age onward until they were 19.5 month-old. At 8, 14 and 20 months of age, they were sacrificed and serum and retinal iron levels were detected by HPLC. Oxidative stress was analyzed by TBARS method. The retinas were examined for cell death (TUNEL), histology (electron microscopy) and the expressions of transferrin, transferrin receptor-1 [TFR-1], H- and L-ferritin. In control animals, at any age, there was no difference in the serum and retinal iron levels, but the latter increased significantly in 14- and 20 month-old iron-fed rats, indicating that retinal iron accumulation proceeds with progression of aging (>14 months). The serum and retinal TBARS levels increased significantly with progression of aging in experimental but not in control rats. There was significant damage to choriocapillaris, accumulation of phagosomes in retinal pigment epithelium and increased incidence of TUNEL+ cells in outer nuclear layer and vacuolation in inner nuclear layer (INL) of 20 month-aged experimental rats, compared to those in age-matched controls. Vacuolations in INL could indicate a long-term effect of iron accumulation in the inner retina. These events paralleled the increased expression of ferritins and transferrin and a decrease in the expression of TFR-1 in iron-fed rats with aging, thereby maintaining iron homeostasis in the retina. As some of these changes mimic with those happening in eyes with AMD, this model can be utilized to understand iron-induced pathophysiological changes in AMD.