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  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.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  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. Testing and isolating SARS-CoV-2-positive cases has been one of the main strategies for controlling the rise and spread of the coronavirus pandemic that has affected about 157 million people and 3 million deaths all over the world . Like all other viruses, the SARS-CoV-2 genome has undergone several mutations during its passage through the human host, and the positive or negative impact of most of them on virus transmission and immune escape are still under investigation .In December 2020, health authorities in the United Kingdom had reported the emergence of a new SARS-CoV-2 variant (lineage B.1.1.7) that is phylogenetically distinct from other circulating strains in the region. With 23 mutations, this variant had rapidly replaced former SARS-CoV-2 lineages occurring in the region and has been established to have greater transmissibility through modeling and clinical correlation studies . SubsequAs air travel has resumed in most countries post lockdown, infected individuals harboring these mutations have been detected in countries far away from their origin, generating concerns about greater disease incidence unless these cases are recognized and isolated. That is particularly important as vaccine roll-out has been initiated in several countries, and the studies about vaccine efficacy against the new variants are only in their infancy.Among the diagnostic methods that have been employed for identifying coronavirus infected cases, qRT-PCR has been the most widely adopted nucleic-acid-based test due to its ability to identify even low copy numbers of viral RNA in patient samples and has been considered as a gold standard in diagnosis . HoweverFrancisella novicida (FnCas9), we have recently reported the development of the FELUDA (FnCas9 Editor Linked Uniform Detection Assay) platform for SARS-CoV-2 diagnosis with similar accuracy as the gold standard qPCR test  for identifying CoV2 signatures in patient samples . Using tPCR test . The assPCR test , we specThe study was designed to evaluate the efficacy of RAY on left-over patient samples. The intent of the study was to develop a robust CRISPR diagnostic that can perform with high accuracy for variant detection at significantly low cost and time taken for detection as compared to sequencing. For the N501Y detection using RAY, SARS-CoV-2 and control RNA samples were received from the diagnostic laboratory at CSIR-Institute of Genomics and Integrative Biology.A list of all oligos (Merck) used in the study can be found in Escherichia coli Rosetta 2 (DE3) (Novagen). Rosetta 2 (DE3) cells were cultured at 37°C in LB medium (with 50 mg/ml Kanamycin) and induced when OD600 reached 0.6, using 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG). After an overnight culture at 18°C, E. coli cells were harvested by centrifugation and resuspended in a lysis buffer  glycerol supplemented with 1X PIC (Roche) containing 100 µg/ml lysozyme. Subsequent cell lysis by sonication, the lysate was put through to Ni-NTA beads (Roche). The eluted protein was further purified by size-exclusion chromatography on HiLoad Superdex 200 16/60 column  in a buffer solution with 20 mM HEPES pH 7.5, 150 mM KCl, 10% glycerol, 1 mM DTT. The purified proteins were quantified by Pierce BCA protein assay kit (Thermo Fisher Scientific) and stored at −80°C until further use.Plasmids containing FnCas9-WT and dFnCas9   sequenceAll 48 VOCs/VOIs within 12 emerging SARS-CoV-2 lineages till May 2021 were taken from the Centers for Disease Control and Prevention report . Furtherhttp://jatayu.igib.res.in).For the benefit of end users with quick design and implementation of the RAY for any targetable SNV/VOC/VOI, we now have also optimized our previously developed web tool JATAYU  , which fHBB gene was used as substrate in in vitro cleavage assays, as optimized in our previous studies (2) at 37°C for 10 min, the cleaved products were visualized on a 2% agarose gel and densitometric quantification of images were done  at 37°C for 10 min, and cleaved products were visualized on a 2% agarose gel.Purified PCR amplicons containing WT and Mutant sequences were used as substrates in  studies . SubstraS gene containing N501Y/T716I/E484K mutations was reverse transcribed and amplified using a single end 5’ biotin-labeled primer. In vitro transcription of sgRNAs/crRNAs was done using MegaScript T7 Transcription kit (ThermoFisher Scientific) following manufacturer’s protocol and purified by NucAway spin column (ThermoFisher Scientific). Chimeric gRNA (crRNA:TracrRNA) was prepared by equally  combining respective crRNAs and synthetic 3'-FAM-labeled TracrRNA in an annealing buffer  by heating at 95°C for 2–5 min and then allowed to cool at room temperature for 15–20 min. RNP complex was prepared by equally mixing  Chimeric gRNA and dead FnCas9 in a buffer  and rested for 10 min at RT. Target biotinylated amplicons were then treated with the RNP complexes for 10 min at 37°C. Finally, 80 µl of Dipstick buffer was added to the mix along with Milenia HybriDetect one lateral flow strip. The strips were allowed to stand in the solution for 2–5 min at room temperature and the result was observed. The strip images can be processed using the TOPSE application that generates background corrected values from the smart phone acquired images of the strip. This application has been previously trained on a large number of CoV-2 samples  gene, JOE (VIC or HEX) channel for E gene, and CY5 channel for internal reference.qRT-PCR was performed using STANDARD M nCoV Real-Time Detection kit (SD Biosensor) as per manufacturer’s protocol. Briefly, per reaction 3 µl of RTase mix and 0.25 µl of Internal Control A was added to 7 µl of the reaction solution. Five µl of each of the negative control, positive control, and patient sample nucleic acid extract was added to the PCR mixture dispensed in each reaction tube. The cycling conditions on the instrument were as follows: Reverse transcription 50°C for 15 min, Initial denaturation 95°C for 1 min, 5 Pre-amplification cycles of 95°C for 5 s; 60°C for 40 s followed by 40 amplification cycles of 95°C for 5 s; 60°C for 40 s. Signal was captured in the FAM channel for the qualitative detection of the new coronavirus (SARS-CoV-2) Individuals who were found to be RT-PCR positive for SARS-CoV-2, were sequenced to determine whether it was a UK variant (20I/501Y.V1) or non-UK variant. With a low number of samples arriving each day in the lab and with quick turnaround time for reporting, Nanopore sequencing was undertaken for most of the samples.In brief, 100 ng total RNA was used for double-stranded cDNA synthesis by using Superscript IV  for first strand cDNA synthesis followed by RNase H digestion of ssRNA and second strand synthesis by DNA polymerase-I large (Klenow) fragment . Double stranded cDNA thus obtained was purified using AMPure XP beads . The SARS-CoV-2 genome was then amplified from 100 ng of the purified cDNA following the ARTIC V3 primer protocol. Sequencing library preparation consisting of End Repair/dA tailing, Native Barcode Ligation, and Adapter Ligation was performed with 200 ng of the multiplexed PCR amplicons according to Oxford Nanopore Technology (ONT) library preparation protocol-PCR tiling of COVID-19 virus (Version: PTC_9096_v109revE_06Feb2020). Sequencing in sets of 24 barcoded samples was performed on MinION Mk1B platform by ONT.https://community.nanoporetech.com) with phred quality cut-off score >7 on GPU-linux accelerated computing machine. Reads having phred quality scores less than seven were discarded to filter the low-quality reads. The resulting demultiplexed fastq were normalized by read length of 300–500 (approximate size of amplicons) for further downstream analysis and aligned to the SARS-CoV-2 reference (MN908947.3) using the aligner Minimap2 v2.17   of the sgRNA with respect to the target . Through distal) . Thus, mTo develop RAY for identifying SARS-CoV-2 variants, we first analyzed the mutations arising in all SARS-CoV-2 lineages reported so far (May 2021) and looked for the possibility of FnCas9-mediated targeting based on the presence of an NGG PAM site in the vicinity. We found that out of the 48 unique SNVs reported across all the VOCs and VOIs reported so far , 19 SNVsN and S gene of SARS-CoV-2 which could detect positive cases with high sensitivity and specificity  mutation can be adapted for COVID-19 detection under laboratory conditions. However, dye-based systems have inherent low sensitivity and cannot be used for routine diagnosis. We reasoned that for RAY to detect and diagnose variants, we need to adapt the detection to a more sensitive readout. In an earlier study, we had reported that using a lateral flow assay, a catalytically inactive (dead) FnCas9 generated by mutating the RuvC and HNH domains can be used to accurately identify the presence of SARS-CoV-2 RNA in patient samples . To enabWT) if the sample is WT and by both sgRNAs (SWT and SN501Y) if the sample contains the N501Y variant  should be several folds higher than wild-type samples . To generate a visually distinctive signal between WT and mutant sample, we performed a single-step reverse transcription PCR to generate a biotin-labeled amplified product that can be detected by a single sgRNA  (We first generated a single amplicon labeled with biotin at one end and performed RAY with both sgRNAs (S SN501Y) . We reas SN501Y) .Next, we tested RAY on RNA extracted from samples of eight qRT-PCR-positive SARS-CoV-2-infected individuals who harbored the N501Y mutation . The samples were sequenced in parallel to detect the presence of N501Y mutation. RAY was able to correctly identify the variant signature in all eight samples that harbored the mutation . HoweverTo improve the performance of the assay for patient samples across a wide range of viral titres, we modified several aspects of the RAY assay. Firstly, we used TOPSE , a smartphone-based application to generate a band intensity score from a lateral flow strip to eliminate bias that can result from visual estimation (Appendix 1). Secondly, we labeled both forward and reverse primers in the assay with 5' Biotin to increase the signal intensity and reduced the amplicon length of the substrate to get a consistent amplification at the end of each PCR run .WT sgRNA-binding site, we modified the assay to perform two simultaneous PCR reactions from the same sample, one generating the N501Y specific amplicon (149 bp), and another corresponding to the SWT sgRNA from the original FELUDA assay (287 bp) (We observed that upon double biotinylation the test band intensity for the 580 bp amplicon was 1.5 times higher than single biotin-labeled amplicon . To incr(287 bp) . The douN501Y sgRNA signal that was at least 5.5 times higher than that of the WT sample up to a Ct value of 34. Upon further dilution, no signal was observed both in RAY or qRT PCR for these samples . We observed that RAY was able to give a clearly discernible S samples . Taken t samples .WT RAY were first performed to confirm SARS-CoV-2 positivity  or the N501Y variants (n = 22) identified by sequencing. For each of these samples, a qRT-PCR and the St values . Thus, Rin vitro synthesized sgRNAs , RAY has limitations in multiplexing several VOC candidates in a single assay. Although longer amplicons can be used to identify multiple mutations that appear close to each other, we observed a drop in sensitivity probably due to reduced PCR efficiency for longer amplicons. Similarly, to increase the throughput of the assay, converting the readout into a fluorescence-based mode might be beneficial.Perhaps, the most imperative area of improvement for RAY is increasing the number of possible mutations that can be targeted. The necessity of an NGG PAM at a fixed position away from the position of the target mutation limits the total number of targets that RAY can identify. This can be significantly expanded by systematically exploring more sites in the sgRNA where a mutation weakens the enzyme:substrate interaction. Alternatively, engineering the enzyme to generate PAM-flexible versions can be considered.Routine sequencing of patient samples for disease surveillance is fraught with issues such as cost, logistics, and turnaround times. A typical deep sequencing cost per sample is several orders of magnitude higher than RAY  to develop nucleic acids detection platforms such as SHERLOCK  and DETECTR . Both SHERLOCK and DETECTR rely on the "collateral effect" – promiscuous cleavage and degradation of neighboring ssRNA and ssDNA by Cas13 and Cas12a. Upon target binding, it will cleave the ssDNA probe. These CRISPR-based detections were used for SARS-CoV-2 detection after then .Authors previously reported that Francisella novicida (FnCas9) could be used for gene editing. In authors' preprint project, they reported the high specificity of FnCas9 to the point mismatch at the specific position of gRNA and there is no "collateral effect" of FnCas9. Based on this discovery, they developed a non-cleavage, affinity-based method for the nucleobase detection with single nucleotide mismatch sensitivity (ref 25).Authors leverage the single nucleotide mismatch sensitivity of FnCAS9 and report herein a non-cleavage, affinity-based SARS-CoV-2 variants detection.Strengths:– This assay provides a simple, rapid way to detect SARS-CoV-2 and its SNV.– Authors demonstrated the detection of SARS-CoV-2 and SAR-S-CoV2 variants (N501Y) in 18 patient samples and validated the samples by sequencing.Weaknesses:– Although authors cite their previous preprint paper , authors didn't describe any sensing mechanism of this assay (RAY). Readers cannot understand this assay by reading this manuscript only. The lack of description in this manuscript strongly affects readers to understand the experiments. Authors assume readers have read another article from the same group and understand the design and mechanism of their "FELUDA" assay. For example, Authors didn't mention that active FnCas9 was used in the experiment in Figure 2a while catalytic inactive FnCas9 was used for SNV detection assay "RAY".– Only N501Y detection was demonstrated in this project. Authors did not demonstrate the generalizability of this assay by demonstration of detecting other SARS-CoV-2 variants. Furthermore, authors mentioned that only 10/32 SNV sites were targetable by FnCas9.– The concept / sensing mechanism of this assay is very similar to the assay reported in the same group (ref 25). They used a lot of results / discovery in the preprint (ref 25) to develop this assay and there are only 4 sets of new data reported in this manuscript.There are several points that may help authors to improve this project."We found that out of the 32 defining SNVs 100 reported across all the three lineages, 10 sites were targetable by FnCas9 "Figure 1B shows the evolution of SARS-CoV-2 virus. How is it related to this statement? Figure 1C shows the position of different mutations in the genome. Why is it important to show the position of the mutations? Is it related to the targetability of FnCas9? Explanation is needed.Authors should explain how they find 10 of 32 single nucleotide variation (SNV) sites are targetable by FnCas9. Why only 10/32 SNV sites are targetable? Authors should explain this result. Without this explanation, readers will not understand how this assay works and how to use this assay to detect other SNV.As only 10 of 32 SNV sites are targetable, authors should discuss the generalizability of this assay."Thirdly, we made modifications at the level of amplicon length, PCR conditions, and RNP concentration to generate the most optimal protocol that can produce a clear discernible signal on a commercially available paper strip"No data is shown about PCR conditions and RNP concentration optimization. Why does the assay not work well when the amplicon length is 541 bp or less?In my view, the combination of the preprint paper (ref 25) and this manuscript will make it a detailed, comprehensive project.There are additional points that need addressing to improve this paper.Only N501Y detection was demonstrated in this paper strip assay. Paper strip detection of other targetable SNV sites could show the generalizability of this assay in specific conditions.This manuscript has a lot of abbreviations, but some of them only appear 1 or 2 times. It makes this manuscript difficult for readers to read.It is confused that Wt, mut and N501Y are all present in Figure 2a. Authors are suggested to label it more clearly to show that it is WT and mutated sgRNA (N501Y)."Finally, we tested RAY on RNA extracted from samples of RT-PCR positive SARS-CoV-2 infected individuals who returned to India from the United Kingdom between December 2020 and January 2021."The travel history of the patient is not necessary for assay validation.Reviewer 2:The authors report the development of a CRISPR diagnostic platform for detecting variants (N501Y and T716I) in SARS-CoV-2. This is based on a technology, recently developed by the same research groups, using a Cas9 ortholog from Francisella novicida (FnCas9) based enzymatic readout for detecting nucleotide sequences. RAY (Rapid variant Assay), detect both the infection as well as the presence of the common N501Y mutation present in the 3 new variants and distinguish it from the parent CoV-2 lineage. The authors also adapt RAY to a lateral flow paper strip assay for low-cost rapid detection. This represents a novel sequencing-free rapid detection system for CoV-2 mutations.Although the paper does have major strengths in principle, the authors have not provided sufficient data and information directly for an end user to adapt this platform to their use. For a stand-alone resource article, authors must provide all the critical information within the text, figures, methods and supplementary figures required to understand the range, specificity, sensitivity, limits and error prone aspects (if any) of the technology.Further information and clarification are required for the following points:Primer design and selection:1. Given that the authors mention that they successfully established that FnCas9 is unable to bind or cleave targets having two mismatches -in the 2nd and 6th position; why were PAMs searched with mismatches in the 16th and 19th position ?2. How many pairs of flanking primers need to be designed for a specific point mutation? What experimental output would demonstrate optimal primer design?3. More details on the amplicons are required.IVC Detection1. What size fragments were generated? Was only one sgRNA used for each N501Y and T716I?In figure 2a it seems like the amount of wild type amplicon is higher or equal to one of the cleaved fragments in the N501Y sample gel (mut lane). Is this due to poor cleavage by the FnCas9? A densitometry analysis would provide better quantification of fragment formations.2. What is the specificity, selectivity for each of these variants? Do altering sgRNA change the specificity? What conditions are necessary for the reaction to go to completion? How does one account for false negatives; is there a low copy number limit for this detection system. No experimental results have been provided for any of this.3. For the reaction between FnCas9 and sample/amplicons what are the ratio ranges; concentrations; volumes to be followed?LFA Detection1. Please elaborate the protocol for "modified single step" RT-PCR.2. The authors mention that they eliminate one of the biotin label primers to reduce the chances of background signal due to non-specific interactions of the unused biotin primers with the streptavidin. The authors should compare and quantitate the output using various methods used in the current work as well as previous FELUDA paper 3. Authors should explain the difference between FnCas9 and dFnCas9 in at least the methods.4. What are the limits of detection of RAY? Is it similar to FELUDA? How does changing sgRNA/ chimeric gRNA effect the detection limit? No experimental results have been provided for this.5. If the only method of detection analysis is TOPSE then the authors must provide more details of how to access/use the app.Reviewer 3:The work by Kumar et al. presents a new diagnostic technique  for detecting SARS-CoV-2 virus infection (responsible for the current COVID-19 pandemic). The researchers use a CRISPR-based method to provide a "quick" assay that can distinguish COVID positive and negative samples. This method is an extension of their previous work on a COVID-19 assay called FELUDA (https://www.medrxiv.org/content/10.1101/2020.09.13.20193581v1). The focus of the current work is to specifically identify COVID-positive samples as well as the particular variant of the viral genome. Typically, the different SARS-CoV-2 variants could differ by a few differences in the genome sequence. This work uses the specificity of the CRISPR reaction: the enzymatic cleavage only occurs for a completely complementary target and not for a target with mutations. The authors developed a lateral flow assay, where the signal corresponding to detection of the viral RNA and/or the mutant is read out as a line on a paper strip (similar to pregnancy strips). Appearance of these lines indicate presence of the virus in the sample. The work used 8 COVID positive samples, 8 positive samples with the mutation N501Y , and 12 COVID negative samples, and identified all the clinical samples accurately.In terms of the scope, the current work demonstrates COVID19 mutation detection with an easy lateral flow-based readout, within ~75 minutes. While the assay has been shown to be able to detect 20 samples accurately, a larger data set might be needed to evaluate the sensitivity of the assay and the range of viral loads it can detect. The past year has seen a number of new techniques for COVID19 diagnosis, all of which will be useful for the still ongoing large-scale testing worldwide. In that context, this assay has practical and immediate use for COVID19 detection if it can be commercialized, especially with the rise of a number of variants of the SARS-CoV-2 virus.I liked reading the paper – it was very clear and crisp, and the data was well presented in both the text and the figures. However, as written, the paper seems to rely heavily on the reader's knowledge of their earlier work on FELUDA. From a reader's point of view, I felt that certain aspects of the assay, methodology and scope of immediate use could be expanded here even if it is a repeat from the authors' FELUDA work, to make this a stand-alone paper.Discussion can be included on the following points. While these are not technical concerns, I think it will be informative for scientific and lay audience to know where the assay stands in terms of immediate impact for COVID19 pandemic.– Metrics of the assay such as sensitivity and specificity (even if referring to previous works).– Scope of the assay: Do the authors intend to use this as a clinical test or at POC? Specific aspects can be discussed for either or both of these scenarios.– For the assay to be useful in POC setting, sample processing is an important step. Assuming samples are collected on swabs, RNA needs to be extracted before RAY can be performed. How can this be brought into the regular workflow for the assay to work at a testing site outside a lab or clinical facility? How will this affect the cost of the assay? The tally of the assay cost excludes RNA extraction. How can such an enzymatic approach be performed in a remote site without a cold chain? Briefly, we request a revised manuscript. It is written such that the reader must already be familiar with a previous paper published by the authors, of which this is an extension to detect variants. The manuscript should be stand alone and include all information about this assay and the method validation result. As the title of this manuscript is "detection of variants on a paper strip", generalizability must be addressed if this assay is able to do that. If not, the authors must discuss how to achieve generality and revise the title to "detection of common SARS-CoV-2 variants". In this scenario, the results can support the title.eLife for the positive and helpful suggestions and for considering that this tool has immediate application in healthcare during the current pandemic. We are now submitting a revised manuscript taking care of the points raised by the reviewers, both in terms of a better description of the technology and its modifications and improvements alongside commenting on the generalizability of the assay in the Discussion section. We accept the suggestion made about the title and have amended the same in the new version.We thank the Senior and Reviewing Editors of Reviewer 1:Researchers from different groups have employed CRISPR-Cas9 protein variants  to develop nucleic acids detection platforms such as SHERLOCK  and DETECTR . Both SHERLOCK and DETECTR rely on the "collateral effect" – promiscuous cleavage and degradation of neighboring ssRNA and ssDNA by Cas13 and Cas12a. Upon target binding, it will cleave the ssDNA probe. These CRISPR-based detections were used for SARS-CoV-2 detection after then .Authors previously reported that Francisella novicida (FnCas9) could be used for gene editing. In authors' preprint project, they reported the high specificity of FnCas9 to the point mismatch at the specific position of gRNA and there is no "collateral effect" of FnCas9. Based on this discovery, they developed a non-cleavage, affinity-based method for the nucleobase detection with single nucleotide mismatch sensitivity (ref 25).Authors leverage the single nucleotide mismatch sensitivity of FnCAS9 and report herein a non-cleavage, affinity-based SARS-CoV-2 variants detection.Strengths:– This assay provides a simple, rapid way to detect SARS-CoV-2 and its SNV.– Authors demonstrated the detection of SARS-CoV-2 and SARS-CoV-2 variants (N501Y) in 18 patient samples and validated the samples by sequencing.Weaknesses:– Although authors cite their previous preprint paper , authors didn't describe any sensing mechanism of this assay (RAY). Readers cannot understand this assay by reading this manuscript only. The lack of description in this manuscript strongly affects readers to understand the experiments. Authors assume readers have read another article from the same group and understand the design and mechanism of their "FELUDA" assay. For example, Authors didn't mention that active FnCas9 was used in the experiment in Figure 2a while catalytic inactive FnCas9 was used for SNV detection assay "RAY".We thank Reviewer 1 for his/her assessment of the assay and appreciating its ability to detect SNVs that constitute the VOCs in the SARS-CoV-2 genome. We understand the concerns raised by the Reviewer about the description of the sensing mechanism of RAY and the lack of clarity about the active/inactive versions of the FnCas9 used in Figure 2a. In the revised version we have made changes reflecting a detailed explanation of the assay and its modifications and included a comprehensive Supplementary Note describing the assay implementation.– Only N501Y detection was demonstrated in this project. Authors did not demonstrate the generalizability of this assay by demonstration of detecting other SARS-CoV-2 variants. Furthermore, authors mentioned that only 10/32 SNV sites were targetable by FnCas9.We thank Reviewer 1 for this point. Although we achieved the design of a RAY strategy for multiple variants, we couldn't validate them all due to time and sample availability constraints . However, we picked three of the reported VOCs in the samples we had access to  and validated RAY on patient RNA samples. We were able to detect all three variants using our assay successfully. We chose to perform intensive validation and standardization with N501Y over a larger sample set owing to its common occurrence across multiple lineages .The Reviewer has rightly mentioned about 10/32 SNV sites that were targetable by FnCas9 in our original submission. Due to the rapid progress and evolution of the SARS-CoV-2 virus, currently (as of May 2021), 13 emerging lineages have been reported . Out of these, 5 lineages have been designated as Variants of Concern (VOCs) and 8 lineages as Variants of Interest (VOIs) by the Centers for Disease Control and Prevention (https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/variant-surveillance/variant-info.html#Interest). We reanalyzed the emerging lineages and found that there are 48 SNVs associated with these VOCs and VOIs and RAY design based on existing parameters can target 19 of these SNVs. Importantly, RAY is able to detect every VOC/VOI through at least one SNV. We represent this data in Supplementary Table 1 and Figure 1 (revised manuscript).Interestingly, N501Y is associated with 3/5 VOCs . Thus, by detecting this variant, RAY can detect whether a sample belongs to one of these VOCs. In the remaining VOCs (B.1.427 and B.1.429) RAY for the SNV W152C SNV can be used in detection. However, this has not been practically validated.– The concept / sensing mechanism of this assay is very similar to the assay reported in the same group (ref 25). They used a lot of results / discovery in the preprint (ref 25) to develop this assay and there are only 4 sets of new data reported in this manuscript.Biosensors and Bioelectronics, 2021), the current manuscript shows the detection of SARS-CoV-2 VOCs on a paper strip, which in our opinion, is both novel and has a high translational impact. Currently, identifying SARS-CoV-2 variants from patient samples exclusively relies on deep sequencing that is both costly and time-consuming. Thus, the value of this manuscript goes beyond simple discovery to a more useful public resource methodology which we hope gets implemented on a larger scale. To enable this paper-strip readout that is robust and performs with high sensitivity and specificity required several rounds of iteration and modification of the original FELUDA protocol and cannot be seen as an extension of the previous work.We thank the Reviewer for this point but would politely disagree. Although the sensing mechanism occurs through FnCas9 and as was described before "Figure 1B shows the evolution of SARS-CoV-2 virus. How is it related to this statement? Figure 1C shows the position of different mutations in the genome. Why is it important to show the position of the mutations? Is it related to the targetability of FnCas9? Explanation is needed.We thank the Reviewer for pointing this out. We agree that Figure 1B does not appropriately correlate with the statement made and has now been replaced in the revised manuscript. We have also explained this better in the manuscript and hope that these modifications will help understand the targeting scope of RAY.Authors should explain how they find 10 of 32 single nucleotide variation (SNV) sites are targetable by FnCas9. Why only 10/32 SNV sites are targetable? Authors should explain this result. Without this explanation, readers will not understand how this assay works and how to use this assay to detect other SNV.As only 10 of 32 SNV sites are targetable, authors should discuss the generalizability of this assay.We thank the Reviewer for this point. The mechanism by which FnCas9 based identification of SNVs has now been explained in detail in the revised manuscript . We have also included a statement about the generalizability of this assay in the discussions section and made an amendment to the manuscript title to reflect this."Thirdly, we made modifications at the level of amplicon length, PCR conditions, and RNP concentration to generate the most optimal protocol that can produce a clear discernible signal on a commercially available paper strip"No data is shown about PCR conditions and RNP concentration optimization. Why does the assay not work well when the amplicon length is 541 bp or less?In my view, the combination of the preprint paper (ref 25) and this manuscript will make it a detailed, comprehensive project.We thank the Reviewer for pointing this out. In the revised manuscript, we have demonstrated the conditions for optimization in Figure 3 and Figure 4. We have also discussed the importance of amplicon length on RAY performance in the manuscript and included a detailed protocol as Supplementary Note 1.There are additional points that need addressing to improve this paper.Only N501Y detection was demonstrated in this paper strip assay. Paper strip detection of other targetable SNV sites could show the generalizability of this assay in specific conditions.We thank the reviewer for pointing this out. We have now demonstrated the detection of 2 more SNVs (E484K and T716I) in addition to N501Y on paper strips to show that several SNVs can be targeted through RAY .This manuscript has a lot of abbreviations, but some of them only appear 1 or 2 times. It makes this manuscript difficult for readers to read.It is confused that Wt, mut and N501Y are all present in Figure 2a. Authors are suggested to label it more clearly to show that it is WT and mutated sgRNA (N501Y)."Finally, we tested RAY on RNA extracted from samples of RT-PCR positive SARS-CoV-2 infected individuals who returned to India from the United Kingdom between December 2020 and January 2021."The travel history of the patient is not necessary for assay validation.We thank the reviewer for mentioning these points. In the revised manuscript, we have taken care to refer to abbreviations judiciously and have removed the travel history information from the main manuscript text as suggested.Reviewer 2:The authors report the development of a CRISPR diagnostic platform for detecting variants (N501Y and T716I) in SARS-CoV-2. This is based on a technology, recently developed by the same research groups, using a Cas9 ortholog from Francisella novicida (FnCas9) based enzymatic readout for detecting nucleotide sequences. RAY (Rapid variant Assay), detect both the infection as well as the presence of the common N501Y mutation present in the 3 new variants and distinguish it from the parent CoV-2 lineage. The authors also adapt RAY to a lateral flow paper strip assay for low-cost rapid detection. This represents a novel sequencing-free rapid detection system for CoV2 mutations.Although the paper does have major strengths in principle, the authors have not provided sufficient data and information directly for an end user to adapt this platform to their use. For a stand-alone resource article, authors must provide all the critical information within the text, figures, methods and supplementary figures required to understand the range, specificity, sensitivity, limits and error prone aspects (if any) of the technology.We are grateful to Reviewer 2 for appreciating the technology and critically evaluating the work. We value the concerns raised for providing detailed information within the text, figures, methods, and supplementary figures for better implementation of the assay by an end-user. We have now addressed each of these points in detail in the revised manuscript.Further information and clarification are required for the following points:Primer design and selection:1. Given that the authors mention that they successfully established that FnCas9 is unable to bind or cleave targets having two mismatches -in the 2nd and 6th position; why were PAMs searched with mismatches in the 16th and 19th position ?2. How many pairs of flanking primers need to be designed for a specific point mutation? What experimental output would demonstrate optimal primer design?3. More details on the amplicons are required.We thank Reviewer 2 for pointing this out. We have addressed these as follows:in vitro cleavage outcomes for both 2 and 6 and 16 and 19 mismatched positions . We apologize for not including this crucial piece of data in the original version.1. The PAM search from the 16th and 19th positions was done based on our observation that the combination of these positions could also allow the FnCas9 to discriminate between its targets. In the revised manuscript, we have shown the 2. A single primer pair of flanking primers are needed for a specific point mutation. The experimental output that demonstrates optimal primer design is the distinct difference in band intensities between the WT and SNV samples. In the revised manuscript, we have explained the methodology for smartphone-based evaluation of band intensities and subsequently calling the samples WT or Mutant.3. We have provided details of the amplicon design in the revised manuscript.IVC Detection1. What size fragments were generated? Was only one sgRNA used for each N501Y and T716I?In figure 2a it seems like the amount of wild type amplicon is higher or equal to one of the cleaved fragments in the N501Y sample gel (mut lane). Is this due to poor cleavage by the FnCas9? A densitometry analysis would provide better quantification of fragment formations.We have indicated the size of the fragments in the revised manuscript . Two different sgRNAs were used for N501Y and T716I correspondingly. The sequences of these sgRNAs are provided in Supplementary Table 4 in the revised manuscript. Densitometric analysis showed 72% cleavage of the mutant substrate and 3% cleavage of the WT substrate for N501Y and 83% and 1% respectively for T716I. Taken together this shows that the mismatch containing sgRNAs are highly specific in distinguishing the two substrates. In the revised manuscript, we have shown the outcome of T716I only as a lateral flow assay for clarity .2. What is the specificity, selectivity for each of these variants? Do altering sgRNA change the specificity? What conditions are necessary for the reaction to go to completion? How does one account for false negatives; is there a low copy number limit for this detection system. No experimental results have been provided for any of this.3. For the reaction between FnCas9 and sample/amplicons what are the ratio ranges; concentrations; volumes to be followed?The specificity and selectivity of these variants are high. In the revised manuscript, we present data from 37 WT and 22 N501Y samples and observe a sensitivity of 86 % and specificity of 97 % on LFA . Since the discrimination of SNVs through RAY follows a design based on introducing mismatches, the possibility of alteration in sgRNA doesn’t arise since the mismatch positions are fixed with reference to the mutation.Biosensors and Bioelectronics, 2021), we had optimized the time required for the FnCas9 based substrate-binding reaction to proceed to completion. In this manuscript, we have adapted the same reaction time for RAY.For both the PCR and the subsequent CRISPR reaction, the presence of components  are in excess (details in Supplementary Note 1), and therefore the reaction proceeds to completion. In our previous study , false-negative results arise when the test band intensity is not high enough for visual/app-based detection. This can happen when the viral load is extremely low or the PCR efficiency is not optimal. Alternatively, a very low-intensity band can also be due to degradation of the CRISPR components in the assay (dFnCas protein or sgRNA).The limit of detection expressed as a corresponding RT-PCR Ct value has now been presented in the revised manuscript .LFA Detection1. Please elaborate the protocol for "modified single step" RT-PCR.We provide a detailed protocol of the modified PCR and LFA steps in Supplementary Note 1.2. The authors mention that they eliminate one of the biotin label primers to reduce the chances of background signal due to non-specific interactions of the unused biotin primers with the streptavidin. The authors should compare and quantitate the output using various methods used in the current work as well as previous FELUDA paper in vitro synthesized sgRNAs over multiple rounds of freeze-thaw. Indeed, the same was observed when we performed RAY with phosphorothioate modified N501Y sgRNAs .We thank Reviewer 2 for pointing this out. We have now addressed all the points raised (single vs. double biotin labels) in Figure 4 of the revised manuscript. In our earlier study, we found that phosphorothioate modified synthetic sgRNAs have better stability as compared to 3. Authors should explain the difference between FnCas9 and dFnCas9 in at least the methods.We express regret for not clarifying this point well in the initial submission. We have now explained the difference clearly in the revised manuscript.4. What are the limits of detection of RAY? Is it similar to FELUDA? How does changing sgRNA/ chimeric gRNA effect the detection limit? No experimental results have been provided for this.Biosensors and Bioelectronics, 2021). For RAY, we performed serial dilutions of the WT and mutant strains (N501Y) and found that it was able to discriminate the two accurately up to a Ct value of 34. We have presented this data in Figure 4E (revised manuscript).We thank Reviewer 2 for raising this point. We would like to explain that RAY is an assay for discriminating between two samples (WT and mutant strains of the virus) while FELUDA is for detecting the viral RNA in a single patient sample. Thus for FELUDA, direct quantification of the limit of detection was possible  for detecting SARS-CoV-2 virus infection (responsible for the current COVID-19 pandemic). The researchers use a CRISPR-based method to provide a "quick" assay that can distinguish COVID positive and negative samples. This method is an extension of their previous work on a COVID-19 assay called FELUDA (https://www.medrxiv.org/content/10.1101/2020.09.13.20193581v1). The focus of the current work is to specifically identify COVID-positive samples as well as the particular variant of the viral genome. Typically, the different SARS-CoV-2 variants could differ by a few differences in the genome sequence. This work uses the specificity of the CRISPR reaction: the enzymatic cleavage only occurs for a completely complementary target and not for a target with mutations. The authors developed a lateral flow assay, where the signal corresponding to detection of the viral RNA and/or the mutant is read out as a line on a paper strip (similar to pregnancy strips). Appearance of these lines indicate presence of the virus in the sample. The work used 8 COVID positive samples, 8 positive samples with the mutation N501Y , and 12 COVID negative samples, and identified all the clinical samples accurately.In terms of the scope, the current work demonstrates COVID19 mutation detection with an easy lateral flow-based readout, within ~75 minutes. While the assay has been shown to be able to detect 20 samples accurately, a larger data set might be needed to evaluate the sensitivity of the assay and the range of viral loads it can detect. The past year has seen a number of new techniques for COVID19 diagnosis, all of which will be useful for the still ongoing large-scale testing worldwide. In that context, this assay has practical and immediate use for COVID19 detection if it can be commercialized, especially with the rise of a number of variants of the SARS-CoV-2 virus.I liked reading the paper – it was very clear and crisp, and the data was well presented in both the text and the figures. However, as written, the paper seems to rely heavily on the reader's knowledge of their earlier work on FELUDA. From a reader's point of view, I felt that certain aspects of the assay, methodology and scope of immediate use could be expanded here even if it is a repeat from the authors' FELUDA work, to make this a stand-alone paper.We thank Reviewer 3 for appreciating our work and commenting that the assay has practical and immediate use for COVID19 detection if it can be commercialized. The prime objective for this study is to develop an assay that can detect variants rapidly bypassing the cost and time associated with sequencing, and we are happy to report that we are in talks with interested parties for the global deployment of this assay.We appreciate the Reviewer’s concerns about expanding more about the assay, methodology, and immediate use. We have now addressed each of these points in the revised version of the manuscript.Discussion can be included on the following points. While these are not technical concerns, I think it will be informative for scientific and lay audience to know where the assay stands in terms of immediate impact for COVID19 pandemic.–Metrics of the assay such as sensitivity and specificity (even if referring to previous works).We thank Reviewer 3 for this point. We have now performed this assay on a larger sample size and have included the sensitivity and specificity metrics of the assay in the revised version of the manuscript .– Scope of the assay: Do the authors intend to use this as a clinical test or at POC? Specific aspects can be discussed for either or both of these scenarios.We intend to use this test for rapid early screening of variant signatures. The pipeline of the RAY assay requires a PCR machine and can therefore be done in clinical labs. It then again can be extended to become a POC test in combination with isothermal amplification. Since the FELUDA protocol has been shown to work with Recombinase Polymerase Amplification (RPA), we foresee that adapting RAY to POC-like settings is possible. As Reviewer 3 suggested, we have now discussed these scenarios in the manuscript.– For the assay to be useful in POC setting, sample processing is an important step. Assuming samples are collected on swabs, RNA needs to be extracted before RAY can be performed. How can this be brought into the regular workflow for the assay to work at a testing site outside a lab or clinical facility? How will this affect the cost of the assay? The tally of the assay cost excludes RNA extraction. How can such an enzymatic approach be performed in a remote site without a cold chain?The Reviewer has correctly raised the point about RNA extraction being required for the assay. Currently, the original version of the assay  is being done in lab settings. Since RNA requires careful handling, we do not foresee how this can be avoided except where an automated system for RNA extraction and subsequent assay steps can be set up. If this is successful, the test can be performed outside a lab or clinical facility.We have not included the cost of RNA extraction since the assay has been tested with samples obtained by both columns, automated as well as column-free extraction methods. Since the costs associated with each vary a lot, we have kept the extraction methods separate from the tally.


We present the first clinical integration of a prototype device based on integrated auto-fluorescence imaging and Raman spectroscopy (Fast Raman device) for intra-operative assessment of surgical margins during Mohs micrographic surgery of basal cell carcinoma (BCC). Fresh skin specimens from 112 patients were used to optimise the tissue pre-processing and the Fast Raman algorithms to enable an analysis of complete Mohs layers within 30 minutes. The optimisation allowed >95% of the resection surface area to be investigated . The Fast Raman device was then used to analyse skin layers excised from the most relevant anatomical sites  and to detect the three main types of BCC . These results suggest that the Fast Raman technique is a promising tool to provide an objective diagnosis “tumour clear yes/no” during Mohs surgery of BCC. This clinical integration study is a key step towards a larger scale diagnosis test accuracy study to reliably determine the sensitivity and specificity in a clinical setting. Mohs miin-situ, few have been utilised ex-vivo, to investigate the resection surface of skin specimens excised in Mohs surgery [et al. reported that BCC was identified with a 73% sensitivity and a 96% specificity in digitally coloured FCM images of 544 fresh specimens from 148 BCC excisions [The efficacy of Mohs surgery may increase if the surgical margins could be analysed faster, more reliably and without requiring tissue processing  While multiple techniques were employed for the investigation of BCC  surgery –8. Fluor surgery ,9,10. It surgery , and a m surgery . Recent  surgery . Peters xcisions .Raman spectroscopy is based on inelastic scattering of light by molecules in tissue and was shown to detect endogenous molecular differences between BCC and healthy skin ,15. WhilRecently, we reported the development of a table-top prototype device based on this technique (Fast Raman device), which indicated promising results in a laboratory setting, when investigating frozen skin specimens . The resIn this study, we integrated the Fast Raman device into the Mohs clinical workflow and obtained proof-of-concept results using fresh tissue specimens from all relevant anatomical sites . We also investigated the ability to detect the three main types of BCC , while varying the analysis times between 20 and 30 minutes.2.2.1Skin tissue specimens (114) were obtained from 112 patients undergoing Mohs surgery at the Nottingham NHS Treatment Centre. Ethical approval was granted by the Health Research Authority (HRA) and Health and Care Research Wales (HCRW) (18/WM/0105) and informed consent was obtained from all recruited patients. Tissue layers were included in the study if their size was smaller than 2 × 2 cm, which is the maximum size that can be measured with the Fast Raman device currently, limited by the size of the tissue cassettes (which have a 2.3 × 2.3 cm fused silica window). If an excised layer was larger than 2 × 2 cm, a sub-section of the layer was cut to fit the cassette, if considered not to intervene with the surgical flow.2.2Supplement 1) and loaded in the Fast Raman tissue cassettes (The Fast Raman device was integrated into the Mohs departments at the Nottingham NHS Treatment Centre (Nottingham) and Erasmus MC (Rotterdam), where it was used to investigate tissue specimens intra-operatively. The specimens were excised, washed for superficial blood (detailed in the assettes  – Step 1assettes  – Step 2assettes  – Step 32.3Supplement 1). In Round 1, a single Raman spectrum was measured in each of these segments. This round was used to identify adipose tissue and then eliminate these segments from any further Raman analysis. In Round 2, the remaining number of sampling points (up to a maximum of 550) were distributed in the segments classified as non-adipose tissue, maintaining a consistent sampling point density within these segments by using the method proposed by Takamori et al. [To achieve a fixed and predictable measurement time , the acquisition of the Raman spectra was staggered in three rounds, aiming to direct a higher density of sampling points for Raman spectroscopy to regions with a higher BCC probability, while maintaining a fixed total number of spectra. The auto-fluorescence image of the tissue was segmented to screen out collagen rich dermis, producing segments in regions that have a high possibility of being BCC-positive . The same set of parameters was used for all Fast Raman maps presented in this study.In order to maximise the number of spectra used to train the classification model for each diagnosis, the Fast Raman maps were generated via a leave one patient out cross-validation (LOPO-CV) algorithm. This algorithm was utilised to optimise the parameters and thresholds used by the analysis algorithm, which were selected to produce the Fast Raman maps that best resemble the results of frozen section histopathology  that is negative of tumour can be considered confidently a tumour-clear layer.Supplement 1). The optimization of the tissue pre-processing (to remove blood) and cassette loading (to increase tissue-cassette window contact) was performed using 83 skin layers from 82 patients. These optimized procedures were then tested using an independent set of 31 layers (30 patients). For this, the instrument software was adapted to automatically calculate and display the percentage of the surface area not analysed by the Raman device due to the presence of superficial blood (yellow) or poor tissue-window contact (blue).Surgical ink and superficial blood were observed to absorb the 405 nm laser light used for auto-fluorescence imaging and to produce high intensity Raman spectral bands, affecting the accuracy of the Raman classification model. To circumvent these issues, tissue inking was delayed until after the Raman analysis and tissue specimens were processed to remove superficial blood (detailed in the In The results show that the fraction of the resection surface area not sampled because of poor contact between the tissue and cassette window ranged between 10.6% (nose section) and 0.18% (forehead section). On average, 2.68% (standard error 0.46%) of the surgical margin was not sampled because of poor contact, with a median of 1.69%. We noted that thicker tissue specimens, which contained predominantly stiffer connective tissue , had a higher proportion of their surface area in poor contact with the cassette window . Tissue sections rich in collagen are known to be challenging to flatten in frozen section histopathology . It has For the 31 test tissue layers, superficial blood covered an average 1.76%  of the total resection area. Tissue specimens which contained more muscle, such as cheeks and eyelids, retained more blood, with an observed coverage of the surface of 4.78% and 2.21%, respectively. On the other hand, skin specimens from forehead and eyebrow had less than 1% area with Raman bands from blood.While processing of adipose tissue for frozen section histopathology can be challenging due to the lower temperatures needed to freeze such tissue, often resulting in excessive tissue trimming , this tiOverall, the Fast Raman device was able to analyse > 95.56% of the actual resection surface area of the 31 test layers. These results indicate that the tissue cassette applies sufficient pressure to ensure good contact between tissue and the cassette window, and the auto-focusing mechanism of the instrument produces high quality auto-fluorescence images and Raman spectra over the entire surface area. The pre-processing of tissue was also effective at removing the majority of superficial blood. The investigated area could be further increased by performing multiple relaxing incisions on the epidermal side of stiffer specimens to allow better contact between the tissue and the cassette window and by implementing an automated, user-invariant method for washing/blotting of the tissue blocks .3.3After the optimisation of the tissue handling and washing (83 skin layers from 82 patients), 31 tissue layers (31 patients) were used to investigate whether the Fast Raman device is suitable for analysis of skin layers excised at different anatomical locations relevant to Mohs surgery: nose, temple, eyelid, cheek, forehead, eyebrow and lip. Typical examples of Fast Raman measurements are presented in Considering that the accuracy of the Raman classification model is not 100%, we observed that approximately 10% of tissue layers had segments classified as BCC, which could not be confirmed by frozen section histopathology . The mea3.4Next, we tested the ability of the Fast Raman device to detect the three most common types of BCC encountered in Mohs surgery: nodular , superfiur would be removed with minimal damage to surrounding tissue for this specimen.Furthermore, the Fast Raman device was also shown to detect small residual superficial BCC, such as in the eyelid specimen shown in 3.5In a previous study using frozen skin specimens, the number of Raman spectra acquired was dependent on the size of the tissue, resulting in a total analysis time that varied between 30 to 70 minutes . As a MoIn order to evaluate the performance of the instrument with an even shorter analysis time, we simulated shorter acquisition times for the Raman spectra by artificially adding Gaussian noise to individual spectra (as reported by other groups ). For thAdjustments to the overhead of the software used to control the device could further reduce the measurement time by ∼2 minutes, resulting in possible <20 minutes measurements per tissue layer. The preliminary results presented here suggest that shortening the analysis time to ∼20 minutes (by reducing the acquisition times of the Raman spectra) would retain the same level of performance as the 30 minute measurements.4.This study reports the first clinical integration of Raman spectroscopy in Mohs surgery as a potential alternative technique for frozen section histology. The Fast Raman device combines auto-fluorescence imaging and Raman spectroscopy in an integrated table-top module that enables analysis of skin tissue specimens without micro-sectioning or staining. Tissue specimens from a wide range of relevant head-and-neck Mohs surgery sites were investigated, and the Fast Raman device was able to correctly detect all sub-types of BCC . The analysis can be completed in a fixed and predictable time (30 minutes), which is faster than typical frozen section histology. Our simulations suggest that the measurement time may be further reduced to 20 minutes, without significant changes in detection performance. Completion of the analysis within 20 minutes would allow, if required, for two Mohs layers to be excised while patients are still under local anaesthetic on the operating table. The results show that typically more than 95% of the resection area is analysed by the Fast Raman device, which includes both the epidermal and deep margin, without requiring tissue trimming. Providing “en-face” maps of the tissue is an important advantage compared to histology, where tissue trimming is often required to obtain flat surfaces for sectioning and staining. The Fast Raman device therefore has the potential to provide important benefits to Mohs surgery by reducing the duration and costs of tissue processing and by decreasing diagnosis subjectivity and inter-user variability. However, a larger scale diagnostic test of accuracy study is required to determine the sensitivity and specificity of the Fast Raman device in a clinical setting.


In prostate cancer, overdiagnosis and overtreatment is a common problem for clinicians. Accurate diagnosis and prognosis are essential to avoid unnecessary biopsy and to increases the effectiveness of treatment. A new, easy-to-use and non-invasive test based on liquid biopsy biomarkers such as Progensa PCA3, MyProstateScore, ExoDx, SelectMDx, PHI, 4K, Stockholm3 and ConfirmMDx have been developed to improve diagnosis, prognosis and to help guide the decision-making process. This article provides an overview of the above-mentioned commercial tests. The performance and financial aspects of the tests have been compared using available studies. Then the application of biomarker tests as an adjunct to multiparametric MRI in the diagnosis, prognosis and monitoring of prostate cancer has been discussed.Prostate cancer (PCa) is the most common cancer in men worldwide. The current gold standard for diagnosing PCa relies on a transrectal ultrasound-guided systematic core needle biopsy indicated after detection changes in a digital rectal examination (DRE) and elevated prostate-specific antigen (PSA) level in the blood serum. PSA is a marker produced by prostate cells, not just cancer cells. Therefore, an elevated PSA level may be associated with other symptoms such as benign prostatic hyperplasia or inflammation of the prostate gland. Due to this marker’s low specificity, a common problem is overdiagnosis, which leads to unnecessary biopsies and overtreatment. This is associated with various treatment complications (such as bleeding or infection) and generates unnecessary costs. Therefore, there is no doubt that the improvement of the current procedure by applying effective, sensitive and specific markers is an urgent need. Several non-invasive, cost-effective, high-accuracy liquid biopsy diagnostic biomarkers such as Progensa PCA3, MyProstateScore ExoDx, SelectMDx, PHI, 4K, Stockholm3 and ConfirmMDx have been developed in recent years. This article compares current knowledge about them and their potential application in clinical practice. Prostate cancer (PCa) is the most common cancer in men and the second most common cause of mortality in this population in the United States, with 191,930 new cases and 33,330 deaths in 2020 ..46].The ExoDx Prostate (IntelliScore)  assesses the exosomal RNA expression of three genes  involved in the initiation and progression of PCa. ExoDx prostate is a test performed from a urine sample that does not require prior DRE testing. Exosomal RNA is derived from exosomes, which are small membrane vesicles secreted by several cell types, including immune and cancer cells . The higThe test scores range from 1 to 100 and a cut-off point of 15.6 indicates men at increased risk of HG PCa (≥GG2) at subsequent biopsy, making the test helpful in validating the need for biopsy in men at risk. The test is recommended for men aged ≥50 years who are in the PSA “grey zone” (2–10 ng/mL) to distinguish a benign  from high-grade PCa (HG2 ≥ 15.6) .A validation study  conducteIn a study published 2 years later , McKiernThese two prospective studies validating over 1000 patients ,61 showeOther work ,61 confiThe clinical utility of ExoDx Prostate was recently evaluated in 1094 patients scheduled for their first biopsy (with PSA 2–10 ng/mL). This first study  of PCa bSelectMDx  is a urine-based test after DRE that measures three biomarkers: DLX1 (progression gene), HOXC6 (cell proliferation gene), KLK3 (reference gene) and clinical risk factors . HOXC6 and DLX1 mRNA levels are assessed to estimate the risk of PCa on biopsy and the presence of high-risk cancer.Men with elevated PSA levels in the “grey zone” (4–10 ng/mL) and/or an abnormal DRE result are subjects for whom an initial biopsy is considered. The result determines whether the patient is at high or low risk of PCa. It supports clinical decision-making and stratifies patients into those who may benefit from biopsy and early cancer detection and others for whom it is better to avoid this invasive procedure and continue with routine screening or active surveillance.In a study , 386 menA study  was perfTo evaluate the clinical utility of SelectMDx, 418 patients who had an initial biopsy were studied. A total of 165 of them were positive. The number of biopsies performed within 3 months of the test was reviewed. For patients with a positive result, 71 patients (43%) were biopsied—27 of these patients were identified as having cancer, including 10 with a grade > 2. During this time, 9 patients with negative SelectMDx test results (3.6%) were biopsied—4 were identified as having cancer—all with a grade ≤ 2. SelectMDx has been shown to have a significant impact on decisions about the frequency and timing of biopsies. When the test was positive, the time period was shorter (median: 2 months) and the number of biopsies was five times higher than when SelectMDx was negative (median: 5 months). The test assisted urologists in their decision-making and is, therefore, a useful tool in daily urological practice [A typical dilemma for the urologist deciding on a repeat prostate biopsy was presented in a case report of two men . Both paIt is unknown what the cut-off values should be and what the diagnostic and prognostic accuracy is for a multi-ethnic population. There is a lack of studies on Asian or African American populations.Serum biomarkers, determined from blood samples, are produced by healthy and abnormal cells. PSA is undoubtedly the most widely studied cancer biomarker, but its clinical utility due to low specificity and specificity raises the need to find a test with better diagnostic values. Three tests that may have a positive impact on clinical practice are described below.The Prostate Health Index , determined from a serum test, includes total PSA (tPSA), free, non-protein-bound PSA (fPSA) and (–2)proPSA (the fPSA isoform resulting from incomplete processing of the PSA precursor).Determination of PHI values is indicated in men with PSA levels in the “grey zone” (4–10 ng /mL) and an unsuspected digital rectal examination (DRE) result , at age The above formula indicates that men with lower fPSA, higher total PSA and (–2)proPSA are at an increased risk of development of clinically significant PCa .This was confirmed by a study  in whichHigh PHI values indicate an increased likelihood of detecting prostate cancer, so when a biopsy  is recommended, consideration should be given to using this less invasive method.The PHI score has a greater diagnostic value than considering each of the indices  separately ,51, imprTeams of researchers Lepor et al.  and LoebDe la Calle et al., based on a multicentre study , showed A study  also fouIn response to these publications, Ehdaie and Carlsson  expresseThe authors of the paper , in respn = 503) and an Asian (n = 1652) population, the use of PHI established the recommended cut-off points for the above ethnic groups. More biopsies were avoided in the Asian group (56% vs. 40%). This study also identified the need to establish differential cut-off points for different ethnic groups. The authors of the publication recommended cut-off points for csPCa: PHI > 40 for European men and PHI > 30 for men of Asian origin. This result is not surprising, as Asians have a four times lower risk of prostate cancer than Europeans.In a study  Table 2Table 2 iTo assess the ability of PHI to detect Gleason grade 2–5 (GGG) PCa in Black men, 158 patients with elevated PSA levels and 135 controls were recruited  Table 2Table 2. Currently, some researchers are considering the use of the PHI density index  in diagnosis to identify csPCa. Tosoian et al.  showed tAnother article  examinedThe 4Kscore  is a test developed to identify HG-PCa in patients with a suspicious DRE or elevated serum PSA. The test measures the levels of four kallikreins (4K): total PSA (tPSA), free PSA (fPSA), intact PSA (iPSA) and serum levels of human kallikrein 2 (hK2). It then compares the values obtained in the algorithm with the patient’s age, DRE and results of previous prostate biopsies. Based on this information, the algorithm generates a percentage probability score to predict HG PCa even years in advance. This assessment allows further management to be determined depending on the outcome of the test and a decision to perform an initial or subsequent biopsy. This test is recommended primarily for men with a genetic family history. However, it can be performed by any man over 35 years of age who wants to assess his personalised risk of disease in the future.The 4K test, although not designed to assess the predicted course of already diagnosed prostate cancer, has also been used in patients with csPCa to identify candidates for more intensive therapy. It has also been used to improve treatment selection and thus increase the chance of cure in patients suspected of having an underestimated malignancy. The 4Kscore provides an estimate of a patient’s risk of developing distant metastases within 10 years.Parekh and colleagues  on a valA comprehensive systematic review  includinIn a study , 43,692 In a study  involvinA case–control study  Table 2Table 2 eA study  conducteA study  aimed atStockholm 3 Model (S3M) combines serum biomarkers . The test also takes into account clinical data  and prostate examination . The S3M available in Sweden, Norway, Denmark and Finland is in clinical use for predicting the risk of aggressive prostate cancer and assessing the need for biopsy. The S3M research team at Karolinska Institute is currently working with two major laboratories in Europe, as well as laboratories in the US and Canada, to introduce the test in additional countries around the world. Additional validation studies have been conducted in Germany, the Netherlands and the UK. Studies on non-Caucasian populations  are also planned. If the S3M is negative, the man has a low or normal risk of prostate cancer and is recommended to be followed up in 6 years. If the test is positive, it is recommended that the man is referred to a urologist. The urologist measures the volume of the prostate gland and carries out a DRE. If the prostate volume and/or the DRE test is abnormal, a biopsy is recommended. Otherwise, a Stockholm3 test in 2 years is recommended.A study involving 59,159 men  comparedA study  describeStudies ,69,70,86In a short report , the autLong-term follow-up of the replacement of PSA (as part of the standard prostate cancer diagnostic procedure) with Stockholm3 in prostate cancer detection in primary care in the Stavanger region of Norway showed that the implementation was beneficial. Compared with PSA, S3M reduced the proportion of clinically insignificant PCa (from 58% to 35%) and the number of biopsies performed (from 29.0% to 20.8%). In addition, it increased the proportion of biopsies positive for csPCa from 42% to 65%. This management may also lead to a reduction in healthcare costs. It has been estimated that direct healthcare costs decreased by 23–28% per male studied .S3M is not suitable for men who have previously been diagnosed with or treated for prostate cancer or who are under follow-up after prostate cancer. It has no proven value for men diagnosed with prostate cancer or who have undergone a biopsy or other examination by a urologist within the last 6 months. It does not replace biopsy in men under active monitoring. This test was not evaluated on men younger than 50 years or older than 70 years and was restricted to an ethnically homogeneous population . The S3M was shown to be superior to prostate-specific antigen (PSA) as a screening tool for prostate cancer in all men aged 50–70 years. Furthermore, the S3M test can be performed in cases where the PSA value is > 1.5. The S3M has been shown to be superior in detecting, now often overlooked, aggressive cancer in men with PSA levels of 1.5–3 ng/mL. The S3M may reduce unnecessary biopsies without compromising the ability to diagnose prostate cancer with a Gleason score of at least 7.Tissue biomarkers analyse changes in nucleic acid expression and composition of tissue collected during needle core biopsy of the prostate. The main concept is to detect changes in the histologically normal field neighbouring prostate cancer. This helps to verify whether the patient requires an additional biopsy.ConfirmMDx  is a tissue-based epigenetic assay that uses methylation-specific PCR (MSP) to analyse three prostate-cancer-related changes in DNA methylation patterns of suppressor genes  in biopsy tissue . All these biomarkers were isolated from biopsy-positive tissue.ConfirmMDx is a molecular test clinically validated for predicting prostate cancer risk in men who have had a traditional thick-needle prostate biopsy that did not reveal the presence of cancer cells in collected histopathological material. In many cases, prostate biopsy results are falsely negative. The biopsy specimen may not be cancerous, and the histopathological result will not reveal the presence of cancer. However, due to the “halo effect”, tissue with a normal morphological appearance will show epigenetic changes, indicating the presence of cancer. Using the test, histopathological material that has already been taken—during a prostate biopsy—can be re-examined in a detailed epigenetic analysis quantifying the level of methylation of promoter regions of three genes in benign prostate tissue, assessing with high accuracy the presence of cancer cells in neighbouring areas.ConfirmMDx offers the opportunity to avoid unnecessary repeat biopsies. It allows a decision to be made on whether to include (rule-in) or exclude (rule-out) therapy. High-risk men with a previously negative biopsy may have undetected cancer after the test. Such patients with a previous “false negative” biopsy result should be included for repeat biopsy and appropriate treatment.It also allows low-risk men to be excluded from repeat biopsies, which protects the patient from unnecessary stress and possible complications and reduces healthcare costs. This test increases the negative predictive value.In the MATLOC study  involvinThis was confirmed in the multicentre DOCUMENT study , which vVan Neste et al.  conducteA population of 211 African American men undergoing repeat biopsy was studied to compare the accuracy of predicting repeat biopsy outcomes with previous studies conducted in predominantly Caucasian populations  Table 2Table 2. Wojno et al.  in 2014 A later study  in 2019 ConfirmMDx enables a higher degree of accuracy  and has clinical, financial  and healA prostate MRI costs between 500 and 2500 USD in the United States, depending on whether the patient is insured. Approximately 1 million American men are currently referred for prostate biopsy each year. If all of these men underwent an MRI instead, costs could reach 3 billion USD per year.In a paper  addressiUncomplicated biopsies cost about 1750 USD, those with one complication were already more expensive at 4060 USD, and for patients requiring hospital admission, the cost was as high as 13,840 USD (average cost was 2020 USD). The most common complication of biopsy is bleeding and infection, which can be prevented using biomarker tests from urine or blood. The cost of tests based on these is higher than the commonly used PSA but lower than biopsy, which makes it a cost-effective option.In a paper , SanthiaIn an economic evaluation, Nicholson et al. , compariReports  on SelecThis was confirmed in a study by Govers et al.  on a popBased on studies ,95,96, iThe publication  focused The implementation of ConfirmMDx created a hypothetical commercial health plan in which direct costs were calculated over a 1-year horizon using 2013 Medicare fee-for-service rates. The study concluded that the net cost of a commercial health plan with 1 million members would be reduced by approximately 500,000 USD if patients with histopathologically negative biopsies were screened using an epigenetic test to distinguish between patients who should undergo repeat biopsies and those who should not. The use of this genetic test may reduce healthcare costs and improve clinical management.STHLM3 is not a commercially available test, for which reason its price is unknown. However, it is expected to be similar to other biomarkers currently available (>224 USD). These tests are more expensive than the common PSA, but are more reliable and can be performed less frequently due to their better diagnostic value. It also avoids biopsies, reduces overdiagnosis and allows a treatment plan to be customised to the patient and thus also reduces costs.The National Comprehensive Cancer Network (NCCN) guidelines—version 2.2020—recommend considering the use of biomarkers for the early detection of prostate cancer, indicating that the specificity of screening can be improved in assessing the indication for biopsy (Grade C recommendation). They indicate the possibility of using the Prostate Health Index (PHI), SelectMDx, 4Kscore and ExoDx to assess the likelihood of high-grade cancer .The NCCN guidelines also address post-biopsy management. They indicate the possibility of using tests to improve specificity in high-risk patients despite a negative prostate biopsy result: 4Kscore, PHI, percentage free PSA, PCA3 and ConfirmMDx (included/added from 2020). The recommendations for the management of benign biopsy results themselves have changed “PSA and DRE 6–24 months apart and consideration of per cent free PSA, 4Kscore, PHI, PCA3 or ConfirmMDx and/or mpMRI and/or improved prostate biopsy techniques. Repeat prostate biopsy, depending on risk”. However, the guidelines note that the extent to which tests are validated in different populations varies and that it is unclear what the optimal combination of tests with MRI would be. In the current NCCN guidelines, MPS is listed as a biomarker requiring additional testing.The EAU gives a strong recommendation for the use of risk-calculators and imaging in asymptomatic men with PSA levels of 2–10 ng/mL, while giving a weak recommendation (strength rating—weak) for urine and blood biomarkers to avoid biopsy .The FDA has approved PCA3 and PHI. ExoDx received FDA Breakthrough Design recognition in June 2019. SelectMDx has not been reviewed by the FDA due to the agency determining that such approval is not necessary but includes CAP (College of American Pathologists) and CLIA  accreditations.ConfirmMDx and 4Kscore do not have FDA recommendations but are accredited by CAP and CLIA.Multiparametric magnetic resonance imaging (mpMRI) is a promising new tool for the diagnosis, prognosis and monitoring of PCa.The European Society of Urology guidelines on PCa recommends the use of mpMRI before prostate biopsy in previously untreated patients with suspected PCa .In addition to the high sensitivity of mpMRI in detecting hg-PCa, mpMRI also has disadvantages, i.e., low specificity, high cost, the need for expensive, specialised equipment, low sensitivity in predicting the presence of extra-urethral expansion and the requirement for an expert review. Current research focuses on comparing biomarker tests with mpMRI and also on the extent to which they can complement each other.p < 0.01) and between PI-RADS 4 and 5. The SelectMDx score was better than the PCA3 score in predicting outcome for suspected PCa on mpMRI (AUC = 0.83 for SelectMDx versus 0.65 for PCA3), suggesting the possibility of using the SelectMDx test to stratify patients for mpMRI.A study  on 172 mThe combination of 4KScore (AUC = 0.70) with mpMRI (AUC = 0.74) resulted in a prognostic improvement (AUC = 0.82 for 4KScore and mpMRI combined) in detecting aggressive PCa ,98.In a recent article , the autIn another study , 266 menPHI, total PSA, PSAD and the ability of mpMRI to identify csPCa were compared in a group of 395 men . In deteAdding PHI to mpMRI leads to increased predictive accuracy of csPCa and a reduction of up to 50% in unnecessary biopsies (for men with PI-RADS 3–5 and PHI ≥ 30). Moreover, combination AUC outperforms PHI and mpMRI alone .PCA3 reduces prostate biopsy rates in men undergoing repeat biopsy, but there is still no consensus on the cut-off value.As PCA3 increases with cancer aggressiveness, tests based on it—Progensa PCA3, MiPS and ExoDx—show the ability to distinguish between cancers with high and low Gleason scores, indicating high utility in therapeutic decision-making.As ExoDx uses an algorithm independent of PSA and its derivatives, clinical factors (features) and standard of care (SOC), it is feasible (in the US) to perform at home. The patient takes a sample, hands it over to a courier and then discusses the result with the doctor via telehealth. This novelty (ExoDx Prostate At-Home Collection) seems particularly useful in times of coronavirus pandemics and for people living far from medical care.PHI is significantly better than SelectMDx in diagnosing any PCa, while SelectMDx is significantly better than PHI in diagnosing csPCa.The 4Kscore assesses the risk of detecting HG PCa if a biopsy is performed. It has been shown to have a better detection rate for HG PCa than the modified PCPTRC and SOC. In addition, 4Kscore can predict HG PCa even years in advance and assess the risk of distant metastasis, e.g., in genetically burdened men. It thus helps to non-invasively avoid prostate biopsy for men in whom it is not necessary and identifies men at higher risk for whom an early intervention is beneficial.Hendriks  and collIn a study  on 531 mRusso et al. indicated in their systematic review  the highFurthermore, PHI should not be interpreted as absolute proof of the presence or absence of prostate cancer. Elevated PSA and PHI can be observed not only in patients with prostate cancer but also with benign diseases. PHI results should be interpreted taking into account clinical factors or family history, and individual clinical decisions should be made based on them.Vedder et al.  added PCAdditionally, the previously mentioned study  confirmeIn addition, it is important to remember that drugs such as 5-alpha-reductase inhibitors: finasteride, dutasteride and anti-androgen therapy can affect the levels of PSA and other biomarkers. Such medications should be discontinued for at least 6 months prior to the study. Samples for the test should be taken when the clinician is satisfied that the prostate tissue has recovered, normally no less than 6 months after the date of the last biopsy or any other prostate procedure. The impact of these procedures on the performance of the test has not yet been assessed.Recently, molecular characterisation of PCa has become increasingly important, and a wide range of biomarker-based liquid biopsy tests are commercially available to assist urologists in clinical decision-making. The prostate cancer liquid biopsy biomarkers listed above have a high NPV and therefore help prevent unnecessary biopsies. As mentioned earlier, numerous publications ,17,18 haFrom a clinical point of view, it is critical to identify assays for the early detection of aggressive PCa subtype when it can still be treated effectively. Recent years have led to the development of totally non-invasive tests i.e., (ExoDx Prostate At-Home Collection) where first catch, nondigital rectal examination urine specimens appeared helpful in identifying aggressive (Gleason score 7–10) PCa in a racially diverse patient cohort. Similarly, the four-kallikrein panel showed effectiveness in identifying aggressive PCa in a multiethnic population.It seems that in the near future, molecular biomarkers, clinical and histopathological features and diagnostic imaging will have to be used in a complementary rather than a competitive manner to ensure the best possible selection of patients for mpMRI and eventual biopsy.


WDR36 is one of a number of genes implicated in the pathogenesis of adult-onset primary open angle glaucoma (POAG). Here we describe in detail the phenotype of a patient with pathogenic variation in WDR36 who presented with a protracted history of central vision loss. On exam visual acuities were at 20/100 level, had a tritan color defect and showed central arcuate visual field defects on visual field testing. Enlarged cup-to-disk ratios with normal intraocular pressures were associated with severe thinning of the ganglion cell layer (GCL) and retinal nerve fiber layer consistent with a clinical diagnosis of normal tension glaucoma. Full-field electroretinograms revealed a severe inner retinal dysfunction with reduced amplitudes and remarkably delayed timings of the b-wave, but preserved photoreceptor (a-wave) function. The pattern described herein recapitulates some of the findings of an animal model of WDR36-associated POAG and suggests a mechanism of disease that involves a retina-wide inner retinal dysfunction and neurodegeneration beyond the GCL. Further detailed structural and functional characterizations of patients with a pathogenic variant in the WDR36 gene are required to confirm these findings.  Glaucoma, a chronic progressive optic neuropathy, currently affects 70 million people worldwide with 10% of affected individuals rendered irreversibly blind . ClinicaCYP1B1 have been important clues to address the etiology of glaucoma. Although, over the past 10 years, it is recognized that these variations account for less than 10% of patients presenting with disease [Genetics is considered to play an influential role in understanding the fundamental causes of this very common neurodegenerative disease. Many of the more previously and extensively studied genes such as myocilin, optineurin, and  disease . This ha disease ,9. While disease .LHON or OPA1.A 70-year-old man with a medical history significant for Parkinsonism, who was a non-smoker, minimal drinker with a 10-year history of insidious, progressive, bilateral painless central vision loss. His symptoms began with a “fog” over his vision. He was an avid golfer and described the onset of his symptoms as “being unable to track a golf ball to its final location” after hitting a line drive. His symptoms were “bothersome” but stable, until a year prior to presentation to us, he began to experience difficultly seeing at night, particularly as it related to driving. He also stopped golfing due to progressive visual impairment. His symptoms prompted neuro-ophthalmic evaluation resulting in a diagnosis of an optic neuropathy due to the appearance of his nerves. He had an unremarkable brain and orbital MRIs. He also had a normal comprehensive retinal examination with cupped optic nerves in both eyes but that was otherwise unremarkable. He did not note a family history of consanguinity, however had an extensive family history of profound vision loss in two maternal aunts and three cousins with severe blindness striking as early as 55 years of age. At the time of his initial examination at age 61, his best corrected visual acuity was 20/20 in both eyes. IOPs were in low teens in both eyes. Gonioscopic examination revealed an anterior angle open to scleral spur in both eyes. There was no afferent pupillary defect and color vision in both eyes was normal. His optic nerves were reported as cupped and pale. He had a normal brain MRI and testing for nutritional or toxic optic neuropathies and genetic testing for optic neuropathies . A comprDue to a progressive and insidious central vision loss he sought a second opinion with us at 69 years of age. On examination his best corrected visual acuity was 20/125 in the right eye and 20/50 in the left eye. Fundus exam revealed cupped and pale nerves in both eyes .There were few small depigmented round lesions in the parafovea of both eyes, but otherwise an unremarkable retinal exam, including normal vasculature and retinal periphery. Spectral domain optical coherence tomography (SD-OCT) revealed severe thinning of the retinal nerve fiber layer (RNFL) in the superior and inferior sectors (greatest inferiorly) of the nerve corresponding to a thinned neuro-retinal rim along the same regions and a large (~0.7) cup-to-disk ratios A. The RNWDR36. A central SD-OCT was performed to explore the structural correlate to these retina-wide functional abnormalities  is a complex disease resulting in a characteristic degeneration of the optic nerve through retinal ganglion cell death. Its phenotype results in characteristic damage to axons in an arcuate pattern that results in functional changes in visual fields. While elevation in intraocular pressure is not a prerequisite for the diagnosis of glaucoma, but is the primary risk factor for the progression of the disease . Aside fIn humans, the WDR36 gene has shown varying levels of correlation with POAG diagnosis and severity. Monemi et al. (2005) found a locus for POAG on 5q with 4 variants in the WDR36 gene among 17 unrelated POAG patients, 11/17 with high pressure and 6/17 with low-pressure glaucoma  with residues conserved between WDR 36 orthologs in mouse, rat, dog chimp and human . Monemi Studies have continued to demonstrate equivocal effects with some showing a lack of clear effect in particular populations ,30,31,32WDR36 variants and the genetic contribution to the glaucoma phenotype we present a case of a patient with a clear glaucomatous optic neuropathy confirmed by GCL losses. Interestingly, our patient showed obvious inner retinal functional abnormalities that may be similar to the abnormalities reported in a murine model of the WDR36-associated disease. A search for a similar structural and functional phenotype in other patients as well as the segregation analysis of the phenotype in families with similar molecular defects are needed to confirm the pathogenicity of WDR36 variants in similar situations. Genetic studies will prove helpful in unmasking critical molecular mechanisms that will add in staging, predicting progression, and developing personalized therapies for this debilitating disease. While advancing to this specialized and potentially beneficial areas of treatment, the prevalence of a genetic variants studied for treatment is both population and phenotype dependent and should be considered when developing genetic studies.Although there is some conflict in the literature regarding the role of