key: cord-0977814-if5jdi5t
authors: Azhar, M.; Phutela, R.; Kumar, M.; Ansari, A. H.; Rauthan, R.; Gulati, S.; Sharma, N.; Sinha, D.; Sharma, S.; Singh, S.; Acharya, S.; Paul, D.; Kathpalia, P.; Aich, M.; Sehgal, P.; Ranjan, G.; Bhoyar, R. C.; Indian CoV2 Genomics amp Genetic Epidemiology Consortium,; Singhal, K.; Lad, H.; Patra, P. K.; Makharia, G.; Chandak, G. R.; Pesala, B.; Chakraborty, D.; Maiti, S.
title: Rapid, accurate, nucleobase detection using FnCas9
date: 2020-09-14
journal: nan
DOI: 10.1101/2020.09.13.20193581
sha: e1860a0471650ea494f3a951b51b59532fe42889
doc_id: 977814
cord_uid: if5jdi5t

Rapid detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for accelerated clinical prognosis as has been witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are difficult to implement in settings with limited resources necessitating the development of accurate alternative testing strategies that perform robustly. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a direct Cas9 based enzymatic readout for detecting nucleotide sequences and identifying nucleobase identity without the requirement of trans-cleavage activity of reporter molecules. We demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs) including heterozygous carriers of a mutation and present a simple design strategy in the form of a web-tool, JATAYU, for its implementation. FELUDA is semi quantitative, can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications such as infectious disease outbreaks like COVID-19. Using a lateral flow readout within 1h, FELUDA shows 100% sensitivity and 97% specificity across all range of viral loads in clinical samples. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection at home.

The rise of CRISPR Cas9 based approaches for biosensing nucleic acids has opened up a broad diagnostic portfolio for CRISPR products beyond their standard genome editing abilities 1,2 . In recent times, CRISPR components have been successfully used to detect a wide variety of nucleic acid targets such as those obtained from pathogenic microorganisms or disease-causing mutations from various biological specimens [3] [4] [5] [6] [7] [8] [9] [10] . At the heart of such a detection procedure lies the property of CRISPR proteins to accurately bind to target DNA or RNA, undergo conformational changes leading to cleavage of targets generating a reporter-based signal outcome [11] [12] [13] [14] [15] . To enable such a detection mechanism to be safe, sensitive and All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 14, 2020. . https://doi.org/10.1101/2020.09.13.20193581 doi: medRxiv preprint reproducible across a large variety of targets, the accuracy of DNA interrogation and subsequent enzyme activity is extremely critical, particularly when clinical decisions are to be made based on these results 1, 2 .

Current technologies relying on using CRISPR components for nucleic acid detection can sense the identity of the target either through substrate cleavage mediated by an active CRISPR ribonucleoprotein (RNP) complex or by binding through a catalytically inactive RNP complex. Cleavage outcomes are then converted to a reporter-based readout with or without signal amplification 1 . Among the CRISPR proteins that have been used so far, Cas12 and Cas13 have obtained Emergency Usage Authorization (EUA) for diagnostic use during the COVID-19 pandemic [16] [17] . However each of these approaches has its own strengths and limitations that are related to sensitivity to mismatches and ease of design for wide variety of targets. For example, to genotype individuals with high confidence, including careers of a single nucleotide variant (SNV), Cas12 requires individual sgRNA design and optimization for every target 7 which increases the complexity and thus the time taken for design and deployment of a diagnostic test. Importantly, both Cas12/Cas13 unleash a secondary reporter activity upon activation, leading to possible loss of information about starting copy numbers of the target [16] [17] . Taken together, development of a detection pipeline based on a highly specific Cas protein with a direct binding or cleavage based readout can significantly increase the sensitivity of detection and reduce the time and cost of CRISPR based diagnostics (CRISPRDx), This is especially crucial for pointof-care (POC) applications where complex experimentation or setup of reaction components are not feasible.

We have recently reported a Cas9 ortholog from Francisella novicida (FnCas9) showing very high mismatch sensitivity both under in vitro and in vivo conditions [18] [19] [20] . This is based on its negligible binding affinity to substrates that harbor mismatches, a property that is distinct from engineered Cas proteins showing similar high specificity 21 . We reasoned that FnCas9 mediated DNA interrogation and subsequent cleavage can both be adapted for accurately identifying any single nucleotide variants (SNVs) provided that the fundamental mechanism of discrimination is consistent across all sequences. We name this approach FnCas9 Editor Linked Uniform Detection Assay (FELUDA) and demonstrate its utility in various pathological conditions including genetic disorders. Notably, due to its ease of design and implementation for new targets, we were successful in deploying it for a ready-All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. to-use diagnostic kit during the COVID-19 outbreak that has successfully completed regulatory validation in India [22] [23] [24] .

To identify a SNV with high accuracy, we first sought to investigate if FnCas9 can be directed to cleave the wild type (WT) allele at a SNV by placing an additional MM in the sgRNA sequence specific to the SNV (Supplementary Figure 1A) . To test this, we selected sickle cell anemia (SCA), a global autosomal recessive genetic disorder caused by a point mutation (GAG>GTG) [25] [26] . By fixing the position of the SNV and walking along the entire length of the sgRNA, we discovered that two mismatches at the PAM proximal 2 nd and 6 th positions completely abrogated the cleavage of the SNV target while leaving the WT target intact (Supplementary Figure 1B) . We tested FELUDA on DNA cloned from 6 SCA patients and a healthy control and obtained a clearly identifiable signature for SNV in every case (Supplementary Figure 1C) . Importantly, the same design principle for searching and discriminating can be universally applied to other Mendelian SNVs without the need for optimization (Supplementary Figure 1D) . To aid users for quick design and implement FELUDA for a target SNV, we have developed a web tool JATAYU (Junction for Analysis and Target Design for Your FELUDA assay) that incorporates the above features and generates primer sequences for amplicon and sgRNA synthesis (https:// jatayu.igib.res.in, Supplementary Figure 1E ).

We next sought to adapt FELUDA for fluorescent detection. Unlike Cas12/Cas13 based CRISPRDx platforms, FnCas9 is not reported to produce collateral activity on substrates, so FELUDA is not suitable for trans-cleavage signal output. To circumvent this, we envisioned FELUDA as a direct, non-cleavage, affinity-based method of detection which works with single nucleotide mismatch sensitivity. We first investigated if a catalytically inactive dead FnCas9 (dFnCas9) tagged with a fluorophore (GFP) is adept in sensing a point mutation at DNA using Microscale All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 14, 2020. . https://doi.org/10.1101/2020.09.13.20193581 doi: medRxiv preprint 5 Thermophoresis (MST) ( Figure 1A ). We observed that using FELUDA specific sgRNAs (having 2 mismatches with WT), the WT substrate exhibited negligible dFnCas9-GFP binding (K d = 1037.4 nM ± 93.3 nM) while the SCD substrate showed moderately strong binding (K d = 187.2 nM ± 3.4 nM) ( Figure 1A and Supplementary   Figure 2A ). This is consistent with the in vitro cleavage (IVC) outcomes on the two substrates (Supplementary Figure 1C) . Importantly, both Streptococcus pyogenes Cas9 (SpCas9) and its engineered High-Fidelity variant (dSpCas9-HF1-GFP) showed strong binding to WT substrate with sgRNAs containing 2 mismatches (144.9 ± 11.8 nM and 153.6 ± 19.8 nM respectively) establishing that the inherent DNA interrogation properties of FnCas9 are responsible for discriminating single mismatched targets with very high specificity ( Figure 1A) . We then developed a pipeline to adapt FELUDA for an affinity-based fluorescent read-out system, where an amplification step generates biotinylated products that can then be immobilized on magnetic streptavidin beads ( Figure 1B ). Upon incubation with dFnCas9-GFP, enzymatic binding to the substrate leads to loss of fluorescence signal in the supernatant allowing FELUDA to discriminate between SCA and WT samples ( Figure 1B ).

Although sickle cell trait (SCT) individuals are generally non-symptomatic, carrier screening is vital to prevent the spread of SCA in successive generations and is widely employed in SCA control programs in various parts of the world 26 . Since Figure 1C ). All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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We made several improvements to FELUDA for expanding and simplifying its detection spectrum. To detect non-PAM proximal SNVs we installed an in-built PAM site in the amplification step of FELUDA (PAMmer) and successfully validated this approach using 2 SNVs (A2142G and A2143G) present in Helicobacter pylori 23s rRNA gene which confers variable clarithromycin resistance in patients with gastric Figure 3C) . We also show that FELUDA based detection can work robustly across a wide temperature range and up to 3 days post thawing of reaction components (at room temperature). Thus, field studies using FELUDA can be conducted in diverse climatic conditions and reaction components can be successfully used following cold chain transportation (Supplementary Figure   4A -B).

The recent outbreak of Coronavirus disease 19 (COVID-19) due to SARS-CoV-2 virus provided an opportunity to expand the scope of above-mentioned approach of FELUDA and make a difference in the ongoing public health emergency throughout the world. In addition to general social distancing, identification of infected individuals and, screening their contacts for possible quarantine measures is one of the major steps in reducing community transmission of this virus [31] [32] [33] . Although quantitative Real-Time (qRT) PCR is considered a gold standard test for detecting active COVID-19 cases, such tests are expensive, have long turn-around times and require a dedicated qRT-PCR machine, hence is of limited utility in handling an emergency of this scale. We sought to repurpose FELUDA as a lateral flow assay (LFA) for the All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 14, 2020. . https://doi.org/10.1101/2020.09.13.20193581 doi: medRxiv preprint detection of SARS-CoV-2 that is low-cost, does not need complex instrumentation, and is highly accurate in diagnosis. To enable such a diagnosis on commercially available paper strips we enabled the chemistry of capturing RNP-bound biotinylated substrate molecules on a distinct test line of the paper strip using FAM labeled chimeric gRNA ( 

FELUDA detection is semi-quantitative (due to stoichiometric binding of FnCas9 RNP:target) and therefore shows a strong negative correlation between Ct values and signal intensities ( Figure 3A ). This makes it uniquely placed among CRISPRDx platforms to accurately predict the viral load in patient samples with high reproducibility between assays ( Figure 3B preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 14, 2020. 

In this study, we developed a FnCas9 based test for detecting a wide range of pathogenic SNVs and nucleotide sequences. We were able to design a scalable version of the test that is point-of-care, requires minimal equipment and validate the test on a large number of clinical samples. Our results suggest that FELUDA is All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Genotyping carrier individuals with heterozygosity though sequencing-free methods systems is often complicated and requires extensive optimization of assay conditions 43 . The overall design of the assay is based on the high specificity of Table 1 ). Importantly, optimized conditions and highquality PCR reagents are necessary to ensure robust and consistent FELUDA results (Supplementary Figure 6B) .

We uncovered inconsistencies in qRT PCR C t values between samples that were measured before and after freezing (Supplementary Figure 7A-B) . This is relevant All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 14, 2020. The on-body RT-RPA FELUDA prototype combined with extraction-free RNA isolation can potentially bring the benefits of testing to home. Although RT-RPA is rapid and sensitive, it is also prone to aerosol-based contamination necessitating an efficient cold-chain transportation of pre-pipetted components to minimize user handling 45 . It is imperative to test this prototype on a larger number of clinical samples to establish its accuracy.

Taken together, FELUDA is an accurate and low-cost CRISPR based diagnostic assay to detect nucleic acids and variations. A two-gene FELUDA assay for SARS-CoV2 costs ~7 USD (Supplementary Table 2 ). Its single-mismatch sensitivity to nucleic acids expands its application portfolio to a large number of sectors not limited to healthcare. Its ease of design and implementation, as exemplified by its urgent deployment during the COVID-19 health crisis offers immense possibilities for rapid and wide-spread testing that has so far proven to be successful in spreading the progression of the disease in multiple countries.

The study was designed to evaluate the efficacy of FELUDA on clinical samples. The preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

A list of all oligos used in the study can be found in Supplementary Table 3 .

The sequences for the Hbb (WT and SCA) and GFP were PCR amplified and cloned into the TOPO-TA vector (Thermo Fisher Scientific) to be used as target sequence for Cas9 in vitro cleavage assays. 500bp sequences containing SNV for four Mendelian disorders Glanzman, Thrombasthenia, Hemophilia A (Factor VIII deficiency), Glycogen Storage Disease Type I and X-linked myotubular myopathy were ordered as synthetic DNA oligos (GenScript) and cloned into the pUC57 by EcoRV (NEB) to be used as target sequence for Cas9 in vitro cleavage assays.

Similarly, a 500bp sequence flanking two SNVs (A2142G and A2143G) in Helicobacter pylori 23s rRNA gene were ordered as synthetic DNA oligos (GenScript) and cloned in pUC57 by EcoRV (NEB) to be used as target sequence for Cas9 in vitro cleavage assays.

Catalytically inactive FnCas9 double mutants (D11A, H969A) were generated on pET28-His-10-Smt3-FnCas9 plasmid backbone (Acharya, S. et al. Proc. Natl. Acad.

Sci. 116, 20959-20968 (2019)) by QuickChange II site directed mutagenesis kit (Agilent) following manufacturer's protocol with some modifications.

The proteins used in this study were purified as reported previously 18 . Plasmids containing FnCas9-WT, dFnCas9 and dFnCas9-GFP sequences were expressed in All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 14, 2020. 

In vitro transcription for sgRNAs/crRNAs were done using MegaScript T7 Transcription kit (ThermoFisher Scientific) following manufacturer's protocol and purified by NucAway spin column (ThermoFisher Scientific). IVT sgRNAs/crRNAs were stored at -20Ԩ until further use.

Human Genomic DNA was extracted from the blood using the Wizard Genomic DNA Purification kit (Promega) as per the manufacturer's instructions.

Genomic DNA extraction from human saliva, 1ml of saliva was centrifuged at 13000 rpm followed by three washes with 1ml of 1X PBS. After washing, the pellet was lysed with 50µl of 0.2% Triton X100 at 95°C for 5 minutes. Then again centrifuged at 13000 rpm and supernatant was transferred into a fresh vial. A total volume of 1µl supernatant was used in PCR reaction or stored at -20 °C until further use.

Genomic DNA was extracted from the biopsy samples (15-20 mg) of patients infected with Helicobacter Pylori using DNeasy 96 PowerSoil Pro QIAcube HT Kit using Vortex Genei for the sample lysis.

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FnCas9-sgRNA complex (500nM) was prepared in a buffer (20mM HEPES, pH7.5, 150mM KCl, 1mM DTT, 10% glycerol, 10mM MgCl 2 ) and incubated for 10 min at RT.

Putting together RNP complexes along with DNA amplicons, IVC assays were performed at different temperatures ranging from 10°C to 50°C for 30 min. The reaction was then terminated using 1µl of Proteinase K (Ambion) and removing residual RNA by RNase A (Purelink), cleaved products were visualized on agarose gel.

Post thaw FnCas9 for varied time points starting from 0Hrs to 100Hrs, FnCas9-sgRNA complex (500nM) was prepared in a buffer (20mM HEPES, pH7.5, 150mM

KCl, 1mM DTT, 10% glycerol, 10mM MgCl 2 ). To monitor cleavage activity on linearized DNA plasmid, IVC assays were performed for 30 min with/without 10% sucrose. The reaction was then terminated using 1µl of Proteinase K (Ambion) and removing residual RNA by RNase A (Purelink), cleaved products were visualized on agarose gel.

For the binding experiment using Monolith NT. 115 (NanoTemper Technologies GmbH, Munich, Germany), dFnCas9-GFP protein along with 12% Urea-PAGE purified IVT sgRNAs were used. RNP complex (Protein:sgRNA molar ratio,1:1) was prepared at 25 C for 10 min in buffer (20 mM HEPES, pH 7.5, 150mM KCl, 1mM DTT, 10mM MgCl 2 ). Target dsDNA were formed using HPLC purified 30bp ssDNA oligos (Sigma) by incubating them for 5 min at 95 C and then slow cool at 25 C. dsDNA target sequences varying in concentration (ranging from 0.7nM to 25μM) were incubated with RNP complex at 37 C for 60 min in reaction buffer.

NanoTemper standard treated capillaries were used for loading the sample.

Measurements were performed at 25°C using 40% LED power in blue filter (465-490nm excitation wavelength; 500-550nm emission wavelength) and 40% MST power. Data was analysed using NanoTemper analysis software and plotted using OriginPro 8.5 software. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 14, 2020. . https://doi.org/10.1101/2020.09.13.20193581 doi: medRxiv preprint Sanger Sequencing:

The sequencing reaction was carried out using Big dye Terminator v3.1 cycle sequencing kit (ABI, 4337454) in 10μl volume (containing 0.5μl purified DNA, 0.8μl sequencing reaction mix, 2μl 5X dilution buffer and 0.6μl forward/ reverse primer) with the following cycling conditions -3 min at 95°C, 40 cycles of (10 sec at 95°C, 10 sec at 55°C, 4 min at 60°C) and 10 min at 4°C. Subsequently, the PCR product was purified by mixing with 12μl of 125mM EDTA (pH 8.0) and incubating at RT for 5 min.

50μl of absolute ethanol and 2μl of 3M NaOAc (pH 4.8) were then added, incubated at RT for 10 min and centrifuged at 3800rpm for 30 min, followed by invert spin at <300rpm to discard the supernatant. The pellet was washed twice with 100μl of 70% ethanol at 4000rpm for 15 min and supernatant was discarded by invert spin. The pellet was air dried, dissolved in 12μl of Hi-Di formamide (Thermo fisher, 4311320), denatured at 95°C for 5 min followed by snapchill, and linked to ABI 3130xl sequencer. Base calling was carried out using sequencing analysis software (v5.3.1) (ABI, US) and sequence was analyzed using Chromas v2.6.5 (Technelysium, Australia).

ClinVar dataset (version: 20180930) was used to find out disease variation spectrum that can be targeted by FELUDA 46 . SNVs which are situated 2bp upstream of the PAM sequence were extracted. Further, SNVs with valid OMIM ID having Pathogenic effects were filtered. Finally, variations with higher frequency in Indian Population were selected for the validation using customized python script. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 14, 2020. . https://doi.org/10.1101/2020.09.13.20193581 doi: medRxiv preprint JATAYU, web tool to design sgRNAs and primer for the SNV detection. When provided valid genomic DNA sequence with position and type of variation. JATAYU user interface has been created using Bootstrap 4 and jQuery. And works with a customized python-based Flask framework along with genome analysis tools like BWA (Burrows-Wheeler aligner) and bedtools [47] [48] . Working script is available at https://github.com/asgarhussain/JATAYU.

To design, crRNA specific to SARS-COV 2, genome sequences were downloaded from GISAID (5) . crRNAs were designed by searching any 20 nucleotides followed by NGG PAM. Further, to remove non-specific crRNA, off-target analysis was done by mapping them to human host viruses from Influenza Virus Database 37 Table 1 ).

Synthetic genomic Target for N gene was serially diluted (1:10, 7 times) from ~4x10 6 copies/µl to perform FELUDA reaction. Test band intensity was calculated using TOPSE app (Repeated in three independent experiments).

SARS-CoV-2 positive patient sample was titrated (1:10, 8 times), qRT-PCR was performed using STANDARD M nCoV Real-Time Detection kit (SD Biosensor) as per manufacturer's protocol. Briefly, per reaction 3µl of RTase mix and 0.25µl of Internal Control A was added to 7µl of reaction solution. 5µl 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 instrument were as follows: Reverse transcription 50℃ for 15 minute, Initial denaturation 95℃ All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Extraction free detection of RNA from Saliva samples.

Saliva RNA was extracted from 3 patient samples using RNeasy Kit (Qiagen). For extraction free detection, lysis buffer was prepared by adding 0.5% Triton X-100 in 50mM Tris Buffer pH 5. 0.2U/μl RNase Inhibitor (EUROGENTEC) was added to this buffer solution. 200µl of saliva specimen was collected in a 1.5ml eppendorf tube.

100µl of prepared lysis buffer solution was pipetted to the tube and mixed by flicking.

The tube was incubated at 95°C for 5 minutes on a dry bath after and left undisturbed on bench for 10 min.

One step qRT-PCR was performed by adding 1 µl Reverse Transcriptase (Qiagen) to reaction mix with LightCycler® 480 SYBR Green I Master (Roche). ACTB specific primers at a final concentration of 0.2µM. qRT-PCR reactions were performed using 2ul of the kit extracted RNA and lysed supernatant for each patient. The cycling conditions on instrument were as follows: 1 cycle of Reverse transcription at 42℃ for 15 minute, Initial denaturation 95℃ for 5 min followed by 40 amplification cycles of 95℃ for 10 sec; 60℃ for 30 sec; 72℃ for 30. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

The present study was approved by the Ethics Committee, Institute of Genomics and Integrative Biology, New Delhi and Chhattisgarh Institute of Medical Sciences, All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Bilaspur. Two provisional patent applications have been filed in relation to this work.

Mohd. Azhar is currently an employee of TATA Chemicals, India. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this this version posted September 14, 2020. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this this version posted September 14, 2020. . https://doi.org/10.1101/2020.09.13.20193581 doi: medRxiv preprint