WO2024134572A1

WO2024134572A1 - Sequences and methods for crispr-based detection of omicron 5 variant of sars-cov-2

Info

Publication number: WO2024134572A1
Application number: PCT/IB2023/063086
Authority: WO
Inventors: Suruchi SHARMA; Manasa Bagur PRAKASH; Annes SIJI; Vaijayanti GUPTA; Vijay Chandru; Rajeev Kohli; Sunil Arora; Bharat JOBANPUTRA; Aditya SARDA; Pooja Agrawal; Varsha SRIDHAR
Original assignee: Crisprbits Private Ltd
Current assignee: Crisprbits Private Ltd
Priority date: 2022-12-21
Filing date: 2023-12-21
Publication date: 2024-06-27
Anticipated expiration: 2025-06-21

Abstract

The present disclosure provides means to facilitate the detection of SARS-CoV-2 infection. The present disclosure provides CRISPR sgRNA sequence(s) that allows detection SARS-CoV-2 infection. Said sequence(s) of the present disclosure further facilitate identification and differentiation between variants of SARS-CoV-2 such as Omicron and non-Omicron variants of SARS-CoV-2. Further provided herein are associated method(s) that rely on the specifically designed sgRNA sequence(s) and CRISPR-Cas system(s) employing the same to enable detection of presence or absence of SARS-CoV-2 in a sample, along with differentiation between variants of SARS-CoV-2 such as Omicron and non-Omicron variants of SARS-CoV-2.

Description

SEQUENCES AND METHODS FOR CRISPR-BASED DETECTION OF OMICRON VARIANT OF SARS-CoV-2 TECHNICAL FIELD The present disclosure relates to the fields of molecular biology, genetics and disease management. Particularly, the present disclosure provides means to facilitate the detection of SARS-CoV-2 infection. The present disclosure provides CRISPR sgRNA sequence(s) that allows detection of SARS-CoV-2 infection. Said sequence(s) of the present disclosure further facilitate identification and differentiation between variants of SARS-CoV-2 such as Omicron and non-Omicron variants of SARS-CoV-2. Further provided herein are associated method(s) that rely on the specifically designed sgRNA sequence(s) and CRISPR-Cas system(s) employing the same to enable detection of presence or absence of SARS-CoV-2 in a sample, along with differentiation between variants of SARS-CoV-2 such as Omicron and non-Omicron variants of SARS-CoV-2. BACKGROUND OF THE DISCLOSURE Regular testing for timely isolation has been a crucial activity in the management of the SARS- CoV-2 pandemic. Nucleic acid based detection assay, real-time PCR (rtPCR), ubiquitously proved to be the most effective way of testing for the containment of Covid19 pandemic. This encouraged the development of new ways of nucleic acid based detection which are cost-effective, rapid, and instrument agnostic. Similar to all viruses, SARS-CoV-2 accumulates mutations in its genome over time. Some of these mutations may impact various properties of viruses; for example its infectivity, disease severity, response to therapy, and efficacy of vaccines. The impact of these mutations needs detailed and careful investigation. Given the absence of complete knowledge of the severity of a given variant, the policy makers need effective methods and strategies to track the transmission of new SARS-CoV-2 variants. So far, for SARS-CoV-2 variant tracking, most countries have relied on whole genome sequencing of virus from patient samples to understand the evolution of variants as well as for the tracking of variants once they have been sequenced and the signature mutations have been identified. Sequencing is expensive, time-consuming, and requires specialized instruments, and skilled personnel; thus, underlining the need for a reliable, rapid and cost-effective method for variant tracking. There are various cost-effective and rapid assays in published literature for genotyping of mutations. However, these are not being used in practice for tracking of SARS-CoV-2 variants because these methods require extensive design and validation efforts to develop, and hence are not suitable for rapidly evolving viral variant tracking. CRISPR appears to be a promising technology for sequence specific detection of nucleic acids. There have been various reports demonstrating CRISPR based methods for SARS-CoV-2 detection that show the promise of being instrument minimalistic. Some of them have also shown variant detection capabilities, but most of these variant tracking platforms have not shown the variant selectivity at higher viral loads thus limiting their use. Therefore, there exists a need for a simple method or assay that utilizes the efficiency of CRISPR technology to yield easy and accurate diagnosis of SARS-CoV-2 along with variant selectivity, even at relatively high viral loads. SUMMARY OF THE DISCLOSURE Addressing the aforesaid need in the art for means to provide easy and accurate diagnosis of SARS- CoV-2 along with variant selectivity, the present disclosure provides one or more CRISPR sgRNA(s) selected from a group of sequences represented by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4. In some embodiments, SEQ ID Nos. 1 and 3 bind to a target in the gene encoding S protein of SARS-CoV-2; and wherein SEQ ID Nos.2 and 4 bind to a target in the gene encoding N protein of SARS-CoV-2. In some embodiments, SEQ ID Nos.1 and 3 bind to a target in the gene encoding S protein of SARS-CoV-2 represented by SEQ ID Nos.5 and 7, respectively and wherein SEQ ID Nos.2 and 4 bind to a target in the gene encoding S protein of SARS-CoV-2 represented by SEQ ID Nos.6 and 8, respectively. In some embodiments, the S protein target of SEQ ID No. 1 comprises one or more mutations selected from Q493R, G496S, and Q498R; wherein the N protein target of SEQ ID No. 2 comprises mutation NΔ31-33; wherein the S protein target of SEQ ID No. 3 is a non-Omicron sequence from SARS-CoV-2; and wherein the N protein target of SEQ ID No.4 is a non-Omicron sequence from SARS-CoV-2. Further envisaged in the present disclosure is the CRISPR sgRNA(s) as defined above, for use in detecting SARS-CoV-2 in a sample. Also envisaged in the present disclosure is the CRISPR sgRNA(s) as defined above, for use in detecting an Omicron variant of SARS-CoV-2 in a sample; and/or subvariant of the Omicron variant selected from subvariants BA.1, BA.2 and BA.3. The present disclosure further provides a method of detecting SARS-CoV-2 in a sample, comprising contacting the sample with one or more of the CRISPR sgRNA(s) as claimed in claim 1 in presence of Cas12a nuclease or orthologs thereof, a reporter system for trans-cleavage activity of Cas12a or orthologs thereof and optionally, buffer and/or water. In some embodiments, the reporter system is in the format of F-(N)n-Q or Q-(N)n-F; wherein F is a fluorescent reporter molecule selected from a group comprising SYT09, fluorescein, rhodamine, rhodoamine600, R-phycoerythrin, and Texas Red; wherein N is selected from A, G, T, C, rA, rG, rT and rC; wherein Q is a quencher such as but not limited to black hole quencher (BHQ), Iowa black and 4-(dimethylaminoazo)benzene-4-carboxylic acid (DABCYL) and wherein n ranges from about 6 to about 15. In some embodiments, the method is implemented as an in-vitro assay; wherein the in-vitro assay is performed in a multi-well plate, multi-strip tube or individual tube(s); wherein each well comprises a different CRISPR sgRNA along with Cas12a nuclease, reporter system and optionally, buffer and/or water. In some embodiments, the in-vitro assay further employs additional sgRNA(s) against a target in the SARS-CoV-2 genome different from that targeted by any of the SEQ ID No.1, SEQ ID No. 2, SEQ ID No.3 and SEQ ID No.4. In some embodiments, the additional sgRNA targets ORF1ab gene in SARS-CoV-2; wherein the additional sgRNA(s) has sequence(s) represented by SEQ ID Nos.9 and/or 10. In some embodiments, the method detects Omicron variant of SARS-CoV-2 in a sample; and/or wherein the method detects subvariants of the Omicron variant selected from subvariants BA.1, BA.2 and BA.3. In some embodiments, the sample is a biological sample or an environmental sample. In some embodiments, the sample is subjected to amplification prior to contacting with the sgRNA(s); wherein the amplification is performed by RT-PCR; and/or wherein the application is performed using primer(s) selected from a group comprising SEQ ID Nos.11-18. In some embodiments, the method has analytical limit of detection ranging from about 100 copies to about 500 copies. In some embodiments, the method has specificity ranging from about 80% to about 100%; and/or sensitivity ranging from about 80% to about 100%. The present disclosure further provides a kit comprising the CRISPR sgRNA(s) as claimed in claim 1; Cas12a nuclease or orthologs thereof; reporter system for trans cleavage activity of Cas12a nuclease or orthologs thereof; and optionally, water, buffer, additional sgRNA(s) against a target in the SARS-CoV-2 genome different from that targeted by any of the SEQ ID No.1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, the primers as defined in claim 14, reagents for amplification, a multi-well plate or multi-strip tube or individual tube(s), and an instruction manual. In some embodiments, the sgRNAs are contained in the multi-well plate or multi-well plate multi- strip tube or individual tube(s); and wherein the multi-well plate or multi-well plate multi-strip tube or individual tube(s) comprising the sgRNAs constitutes an assay device. BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where: FIG 1. depicts a) Workflow of the OmiCrisp assay that involves two steps: 1. Amplification 2. Detection. b) Results of the trans-cleavage assay in presence of 30 nM DNA target with indicated guide RNA pair. The graph shows that the ability of the guide pair for N and S gene to discriminate Omicron variant from non-Omicron variant is retained at saturating target DNA concentrations that can be achieve in the assay. c) The analytical validation of the OmiCrisp with specified synthetic RNA as input at indicated concentrations. FIG 2. provides results of clinical validation of OmiCrisp_v1. a) Bar graph showing the fluorescence intensity after one hour of trans-cleavage assay with the detection mix containing indicated sgRNA for each sample. The height of the bar represents the mean intensity of the technical duplicates, and the empty circle overlaid on bars indicate the intensity of the individual technical duplicate. b) Tabular summary of the clinical validation, showing the comparison between sequencing and Omcrisp_v1. c) qRT-PCR analysis of the sample identified as SARS- CoV-2 negative by the OmiCrisp_v1. FIG 3. provides results of clinical validation of OmiCrisp_v2. a) Bar graph showing the fluorescence intensity after one hour of the trans-cleavage assay with the detection mix containing the indicated guide RNA for each sample. The height of the bar represents the mean intensity of the technical duplicates, and the empty circle overlaid on bars indicates the intensity of the individual technical duplicate. b) Tabular summary of the clinical validation, showing the comparison between sequencing and Omcrisp_v2. FIG 4. depicts i) Selectivity of S339 guide pair tested using one step RT PCR followed by the Cas12a trans-cleavage assay.5 * 10^5 copies of the indicated RNA as input for the RT-PCR and 5 µL of the RT-PCR product was used as input for trans-cleavage in 50 µL reaction. ii) S679 guide pair selectivity testing a) one step RT PCR followed by the Cas12a trans cleavage assay.5 * 10^5 copies of the indicated RNA as input for the RT-PCR and 5 µL of the RT-PCR was used as input for trans-cleavage in 50 µL reaction. b)The S679 guide pair selectivity tested with 30 nM of purified DNA target as input for the assay. iii) selectivity of a) S493 guide pair b) N gene guide pair tested using trans-cleavage assay done using 30 nM of purified DNA target as input. FIG 5. depicts results of trans-cleavage assay performed in presence of various buffer compositions using DNA as input at various concentrations: low (0.03 nM), medium (0.3 nM), and high (3 nM). FIG.6. depicts results of testing of lower limit of detection in the presence and absence of sewage matrix. FIG.7. depicts results of testing the limit of detection in a mixed sample. FIG.8. depicts results of testing limit of detection in a mixed sample where the variant of interest is at a low fraction (1:5, 1: 25 and 1:100). FIG. 9. depicts results of testing specificity of the SARS-CoV-2 detection guides in samples previously tested negative for SARS-CoV-2. FIG. 10. depicts results of a) testing of sgRNA of the present disclosure on good quality retrospective samples; b) testing of sgRNA of the present disclosure on good, medium and poor quality retrospective positive sample; c) testing of sgRNA of the present disclosure on retrospective positive samples of medium quality in a time frame where Non-Omicron variants were in circulation. DETAILED DESCRIPTION OF THE INVENTION Addressing the aforesaid need pertaining to accurate identification or detection of SARS-CoV-2 infection in a sample, the present disclosure provides specific sgRNA sequences to enable the said detection, a CRISPR-Cas system employing the same as well as associated methods relying on the said sequences to allow accurate detection of SARS-CoV-2, along with specific variant selectivity. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. A number of terms are defined and used throughout the specification with the following definitions provided for convenience. Definitions The term “CRISPR” as used throughout the present disclosure is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats and refers to the conventional CRISPR technology well known to a person skilled in the art. CRISPR (clustered regularly interspaced short palindromic repeats) are short stretches of DNA sequences discovered in the genomes of prokaryotes, derived from invading phages that attacked them previously. They serve as a memory to destroy similar phages during subsequent infections. More recently, exploiting the high precision and accuracy of these genetic scissors, the technology has been optimized to edit genes in vitro or in vivo, for research as well as therapeutic applications, for treating diseases with clear genetic basis. The term “CRISPR-Cas system” or “CRISPR-Cas complex” and obvious variants thereof as referred to in the present disclosure refers to the collection of elements comprising but not limited to a Cas nuclease and a guide-polynucleotide. The terms “CRISPR guide”, “guide sequence”, “guide RNA” and obvious variants thereof as referred to in the present disclosure refer to the guide-polynucleotide of the CRISPR-Cas system, wherein in the present disclosure, the CRISPR guide is designed to recognize the presence of absence of the mutation(s) of interest at the target loci. In some embodiments, the CRISPR guide is designed as a dual crRNA:tracrRNA guide or a single-molecule guide RNA. The term “single guide RNA” or “sgRNA” as used in the present disclosure is in reference to a single RNA molecule that is sufficient to bind to Cas12a and the corresponding DNA target to initiate the trans-cleavage of a single stranded DNA (ssDNA) reporter. The terms “S protein” and “N protein" have been used in reference to the Spike and Nucleocapsid proteins of SARS-CoV-2, respectively. Similarly, the terms “S gene” and “N gene" have been used in reference to the genes encoding the Spike and Nucleocapsid proteins of SARS-CoV-2, respectively. In some embodiments, the target loci in the S gene and N gene have been referred to as “S-protein target” and “N-protein target”, respectively. The term “Omicron” as used herein is in reference to the Omicron variant of SARS-CoV-2. The term “OmiCrisp” in the context of the present disclosure refers to the assay employing the sgRNAs disclosed herein. As used herein, the term “Reference-specific” is used in relation to the sgRNA designed to provide a positive signal if the selected mutation(s) characteristic of the Omicron variant of SARS-CoV-2 is absent. Said feature of the present disclosure may be interchangeably referred to herein as “SRef” or “NRef” depending on the target in the S and N proteins of SARS-CoV-2, respectively. Similarly, the term “Omicron-specific” is used in relation to the sgRNA designed to provide a positive signal if the selected mutation(s) characteristic of the Omicron variant of SARS-CoV-2 is present. Said feature of the present disclosure may be interchangeably referred to herein as “SOm” or “NOm” depending on the target in the S and N proteins of SARS-CoV-2, respectively. As used herein, the term “subject” denotes a mammal. Preferably reference to a “subject” in the present disclosure implies a human subject. As used herein, the term “sample” refers to biological sample from a subject or an environmental/mixed sample such as but not limited to a sewage sample, food sample, surface swab and milk. The sample is preferably in a state that does not impede amplification reaction and/or hybridization reaction. For example, in some embodiments, to use a material obtained from a living body or the environment (essentially in the form of a mixed matrix) as a sample according to the embodiment, the material may have to pre-processed using a certain means. A nucleic acid contained in a sample is called a “sample nucleic acid” or simply “nucleic acid” in the context of the present disclosure. The term “nucleic acid” assumes ordinary meaning of the said term that is conventionally used and well known to a person skilled in the art. It refers to any one or more nucleic acid segments. Out of the sample nucleic acid, a sequence to be amplified by a primer according to the embodiment is called a “template sequence” or a “template”. Whereas the part of the template recognized by the sgRNA is referred to as “target” or obvious equivalents thereof. As used herein the terms “assay device” or “device” as used interchangeably refer to the CRISPR- Cas based assay platform of the present disclosure to which the sample is applied and that provides a detectable signal or readout allowing for detection of presence or absence of SARS-CoV-2 in the sample. “Amplified nucleic acid” or “amplified sample” refers to an amplified product obtained by amplifying the genomic target of interest from nucleic acid isolated from a sample. “Target loci” or “target of interest” refers to the loci of the mutations that are to be detected by the sgRNAs of the present disclosure. “Amplification mix” in the context of the present disclosure refers to the mix of template, dNTPs, primer(s), buffer(s), polymerase enzyme(s) and the amplified DNA (preferably amplified at target loci) obtained at the end of the amplification cycle that the nucleic acids are subjected to, before being subjected to detection by the sgRNAs of the present disclosure. As used herein, a “primer” (primer sequence) is a short oligonucleotide of an appropriate length sufficient to hybridize to a target DNA (e.g., a single-stranded DNA) and allow addition of nucleotide residues thereto, or an oligonucleotide or polynucleotide synthesized therefrom under suitable conditions well known in the art. The primer is designed to have a sequence complementary to the region of the template/target DNA to which the primer hybridizes. As used herein, the term “reporter” or “reporter system” refers to a “label” that can be used to provide a detectable (preferably quantifiable) signal. Reporters may provide signals detectable by fluorescence, luminescence, radioactivity, colorimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. “Activation” of the reporter system refers to the modification of the reporter system in a way that yields a “detectable signal” or “readout”, indicating the presence or absence of the mutation(s) of interest. The term “detection” or “detection event” as used herein refers to subjecting the sample to recognition by a specific guide RNA, wherein each guide RNA is designed to recognize the sequences specific to SARS-CoV-2 or to confirm presence of signature mutation(s) characteristic of the Omicron variant of SARS-CoV-2. In some embodiments, every detection event – confirming either presence or absence of the target signature mutation(s) in the tested sample yields a detectable signal. Thus, “detection”, in the context of the present disclosure, envisages confirmation of presence or absence of a target sequence in a sample. In the context of an assay, separate detection events, in non-limiting embodiments, may occur in separate tubes, wells on the device surface, lateral flow strips, uniquely tagged beads or in multiplexed reactions. The term “negative control” refers to detection events where no sample is added, where signals could arise from random contamination. The phrase “median negative control signal” refers to the median signal value that is calculated after the negative controls for all the guides are pooled. The phrase “Interquartile range” (IQR) refers to the difference between the 75th and 25th percentiles of the signals obtained from the pooled detection negative controls. It shows what range the bulk of the signal values lie in. The phrase “Standard deviation” in the context of the present invention is in line with the established meaning of the said term in statistics and depicts how individual signals are clustered or dispersed around the mean value calculated for the set of signals. The phrase “average signal of a given guide” refers to the average signal observed between different duplicates for a detection event. With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The use of the expression ‘at least’ or ‘at least one’ suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. Numerical ranges stated in the form ‘from x to y’ include the values mentioned and those values that lie within the range of the respective measurement accuracy as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included. The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, +/-5% or less, +/-1% or less, and +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed. As used herein, the terms “include” (any form of “include”, such as “include”), “have” (and “have”), “comprise” etc. any form of “having”, “including” (and any form of “including” such as “including”), “containing”, “comprising” or “comprises” are inclusive and will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps It is to be noted that the term “a” or “an” entity refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein. As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise. Disclosure The present disclosure is directed towards accurate detection of SARS-CoV-2 infection and more specifically towards distinguishing the variant of SARS-CoV-2 that is causative of the infection. In preferred embodiments, said variant is the Omicron variant of SARS-CoV-2. sgRNAs In order to meet the above objective, specifically provided herein are CRISPR sgRNA(s) selected from a group of sequences represented by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4. Said sgRNA sequences, paired with Cas nucleases, may be employed as part of a tailored assay designed to detect the presence or absence of SARS-CoV-2 in a sample, and furthermore, determine the specific variant of SARS-CoV-2 in the sample. The identification and/or differentiation between different variants of SARS-CoV-2 in a sample is based on the detection of signature mutations in the SARS-CoV-2 genome that are characteristic of SARS-CoV-2 variants. In some embodiments, the sgRNA sequences of the present disclosure help determine the presence or absence of the Omicron variant of SARS-CoV-2 in a sample. Thus, confirmation of presence of the Omicron variant of SARS-CoV-2 in a sample is based on the detection of signature mutations in the SARS-CoV-2 genome that are characteristic of the Omicron variant of SARS- CoV-2. In some embodiments, the sgRNAs having sequences represented by SEQ ID No.1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4 are designed against targets in the SARS-CoV-2 genome represented by SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, respectively. In some embodiments, further sgRNAs may be designed against the same targets in the SARS- CoV2 genome, for compatibility with different Cas nucleases. Such sgRNAs and methods employing the same are therefore envisaged in the scope of the present disclosure. Therefore, while embodiments describing the method and kit of the present disclosure in detail are captured in subsequent paragraphs, the present disclosure envisages detection methods employing sgRNAs that recognize and bind targets in the SARS-CoV-2 genome represented by SEQ ID No.5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8. Without departing from the spirit of the present disclosure, further envisaged herein are kits that employ sgRNAs that recognize and bind targets in the SARS-CoV-2 genome represented by SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8 to facilitate the detection of SARS-CoV2 in a sample, optionally along with identification of the variant and sub-variant of SARS-CoV2. CRISPR-Cas complex Further envisaged herein is a CRISPR-Cas complex, comprising sgRNA selected from sequences represented by SEQ ID Nos. 1-4 in conjunction with a Cas nuclease such as but not limited to Cas12a. In a non-limiting embodiment, the Cas nuclease includes but is not limited to Cas12a and its orthologs. In some embodiments, orthologs of Cas12a nuclease may be obtained from organisms such as but not limited to Lachnospiraceae bacterium, Acidaminococcus sp., Francisella novicida and Moraxella bovoculi. Reference to Cas12a throughout the present disclosure envisages the equal possibility of reliance on any of the orthologs of Cas12a as defined above. Functional features of the sgRNA(s) The present disclosure provides 2 pairs of sgRNA sequences, one each directed towards the genes encoding the S and N proteins of SARS-CoV-2. In some embodiments, sgRNAs represented by SEQ ID Nos.1 and 3 bind to a target in the gene encoding S protein of SARS-CoV-2. In a non-limiting embodiment, sgRNAs represented by SEQ ID Nos.1 and 3 bind to a target in the gene encoding S protein of SARS-CoV-2 represented by SEQ ID Nos.5 and 7, respectively. In some embodiments, sgRNAs represented by SEQ ID Nos.2 and 4 bind to a target in the gene encoding N protein of SARS-CoV-2. In a non-limiting embodiment, sgRNAs represented by SEQ ID Nos.2 and 4 bind to a target in the gene encoding S protein of SARS-CoV-2 represented by SEQ ID Nos.6 and 8, respectively. Without intending to be limited by theory, in some embodiments, the above recited sequences find application in distinguishing a specific variant of SARS-CoV-2 in a sample based on the presence of a confirmatory signal rendered by a CRISPR based assay employing the said sequences. In some embodiments, the specific variant is the Omicron variant. In some embodiments, the Omicron variant of SARS-CoV-2 is selected from subvariants BA.1, BA.2 and BA.3, identification of the subvariant being facilitated by the aforesaid sequences. Thus, to facilitate the said identification, provided herein are 2 pairs of sgRNAs, one directed towards the gene encoding S protein and the other towards the gene encoding N protein of SARS- CoV-2 to detect the sequences harboring signature mutations of the Omicron variant of SARS- CoV-2. As mentioned above, SEQ ID Nos.1 and 3 bind to a target in the gene encoding S protein of SARS-CoV-2 while SEQ ID Nos. 2 and 4 bind to a target in the gene encoding N protein of SARS-CoV-2. In the said pairs directed towards genes encoding each of S and N proteins of SARS-CoV-2, respectively, one of the guides of a pair is “Reference-specific” and another one is “Omicron- specific”. The “Reference-specific” sgRNA is designed to give a positive signal if the selected signature mutation is absent, and the “Omicron-specific” sgRNA is designed to give a positive signal if the selected signature mutation is present in the sequence. In some embodiments, the S protein target of SEQ ID No. 1 comprises one or more mutations selected from a group comprising Q493R, G496S, and Q498R. In some embodiments, the S protein target of SEQ ID No.1 comprises at least 2 mutations selected from a group comprising Q493R, G496S, and Q498R. In some embodiments, the S protein target of SEQ ID No. 1 comprises mutations Q493R and Q498R. Said protein target, in a non-limiting embodiment, is characteristic of Omicron BA.2 variant of SARS-CoV-2. In some embodiments, the S protein target of SEQ ID No.1 comprises mutations Q493R, G496S, and Q498R. Accordingly, the Omicron-specific sgRNA represented by SEQ ID No.1 is designed to detect the presence of one or more mutations selected from a group comprising Q493R, G496S, and Q498R. The corresponding reference-specific sgRNA represented by SEQ ID No.3 is designed to give a positive signal if the selected signature mutation(s) are absent. In other words, the corresponding reference-specific sgRNA represented by SEQ ID No.3 is designed to give a positive signal for the non-omicron sequence of SARS-CoV-2 at the position corresponding to the S protein target of SEQ ID No.1. Accordingly, in some embodiments, the S protein target of SEQ ID No.3 is a non- omicron sequence from SARS-CoV-2. Said reference-specific sgRNA represented by SEQ ID No. 3 confirms that the detected SARS-CoV-2 strain is a non-Omicron variant of the virus. In some embodiments, the N protein target of SEQ ID No.2 comprises mutation NΔ31-33. Accordingly, the Omicron-specific sgRNA represented by SEQ ID No.2 is designed to detect the presence of mutation NΔ31-33. The corresponding reference-specific sgRNA represented by SEQ ID No.4 is designed to give a positive signal if the selected signature mutation is absent. In other words, the corresponding reference-specific sgRNA represented by SEQ ID No.4 is designed to give a positive signal for the non-omicron sequence of SARS-CoV-2 at the position corresponding to the N protein target of SEQ ID No.2. Accordingly, in some embodiments, the N protein target of SEQ ID No.4 is a non-omicron sequence from SARS-CoV-2. Said reference-specific sgRNA represented by SEQ ID No. 4 confirms that the detected SARS-CoV-2 strain is a non-Omicron variant of the virus. In some embodiments, the sgRNAs targeting the genes encoding S and N protein(s) or specific loci of interest within the said genes, respectively, may be put to application individually or in combination with each other. Application of the sgRNAs As mentioned above, in line with the objective of the present disclosure, the above recited sequences find application in detecting SARS-CoV-2 in a sample. More specifically, the above recited sequences find application in distinguishing a specific variant of SARS-CoV-2 in a sample. In some embodiments, the specific variant is the Omicron variant. In some embodiments, the Omicron variant of SARS-CoV-2 is selected from subvariants BA.1, BA.2 and BA.3, identification of the subvariant being facilitated by the aforesaid sequences. Accordingly, in some embodiments, provided herein are one or more sgRNA(s) selected from sequences represented by SEQ ID Nos. 1-4 for use in detecting SARS-CoV-2 in a sample; preferably an Omicron variant of SARS-CoV-2. In some embodiments, provided herein are one or more sgRNA(s) selected from sequences represented by SEQ ID Nos.1-4 for use in detecting SARS-CoV-2 in a sample. In preferred embodiments, provided herein are one or more sgRNA(s) represented by SEQ ID Nos. 1-4 for use in detecting Omicron variant of SARS-CoV-2 in a sample. In some embodiments, provided herein are one or more sgRNA(s) selected from sequences represented by SEQ ID Nos.1-4 for use in detecting subvariants BA.1, BA.2 and/or BA.3 of the Omicron variant of SARS-CoV-2 in a sample. In some embodiments, provided herein are at least 2 sgRNA(s) selected from sequences represented by SEQ ID Nos.1-4 for use in detecting subvariants BA.1, BA.2 and/or BA.3 of the Omicron variant of SARS-CoV-2 in a sample. In some embodiments, the said use may employ one or more of the four sgRNA(s) defined above, directed towards gene(s) encoding any one or both of the S and N proteins of SARS-CoV-2 in a sample. In some embodiments, envisaged herein are sgRNAs targeting the gene encoding the S-protein of SARS-CoV-2, represented by SEQ ID Nos. 1 and 3, for use in detecting one or more mutations selected from Q493R, G496S, and Q498R in the S gene. In some embodiments, envisaged herein are sgRNAs targeting the gene encoding the N-protein of SARS-CoV-2, represented by SEQ ID Nos.2 and 4 for use in detecting mutation NΔ31-33 in the N gene, In some embodiments, the said use as defined in the above paragraphs may employ sgRNA pair consisting of “Reference-specific” and “Omicron-specific” sgRNAs targeting the S protein in SARS-CoV-2. Accordingly, in some embodiments, the said use may employ sgRNA pair consisting of sequences represented by SEQ ID Nos. 1 and 3 targeting the S protein in SARS- CoV-2. In some embodiments, the said use as defined in the above paragraphs may employ sgRNA pair consisting of “Reference-specific” and “Omicron-specific” sgRNAs targeting the N protein in SARS-CoV-2. Accordingly, in some embodiments, the said use may employ sgRNA pair consisting of sequences represented by SEQ ID Nos. 2 and 4 targeting the N protein in SARS- CoV-2. In some embodiments, the present disclosure provides sgRNAs represented by SEQ ID Nos.1 and 3 for use in detecting SARS-CoV-2 in a sample; preferably an Omicron variant of SARS-CoV-2. In some embodiments, the Omicron variant of SARS-CoV-2 is selected from subvariants BA.1, BA.2 and BA.3. In a non-limiting embodiment, the BA.2 variant of Omicron does not harbor the G496S mutation. In case of a BA.2 positive sample, the S-protein directed reference-specific as well as the Omicron- specific guides may induce trans-cleavage in the target sequence, potentially confirming the presence of the BA.2 variant of SARS-CoV-2. In some embodiments, the present disclosure provides sgRNAs represented by SEQ ID Nos.2 and 4 for use in detecting SARS-CoV-2 in a sample; preferably an Omicron variant of SARS-CoV-2. In some embodiments, the Omicron variant of SARS-CoV-2 is selected from subvariants BA.1, BA.2 and BA.3. In some embodiments, the present disclosure provides 2 pairs of sgRNAs represented by SEQ ID Nos.1 and 3 and SEQ ID Nos.2 and 4 for use in detecting SARS-CoV-2 in a sample; preferably an Omicron variant of SARS-CoV-2. In some embodiments, the Omicron variant of SARS-CoV- 2 is selected from subvariants BA.1, BA.2 and BA.3. In some embodiments, the aforesaid use may employ one or more additional sgRNAs against a target in the SARS-CoV-2 genome different from that targeted by any of the SEQ ID No.1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4. Without intending to be limited by theory, the additional sgRNA(s) give a positive signal for both Omicron and non-Omicron variants of the SARS-CoV-2, confirming the sample to be SARS-CoV-2 positive, in order to avoid false negative results. In a non-limiting embodiment, the additional sgRNAs target ORF1ab gene in SARS-CoV-2. In a non-limiting embodiment, the additional sgRNAs targeting the ORF1ab gene in SARS-CoV- 2 have sequences represented by SEQ ID No.9 and SEQ ID No.10. Accordingly, in some embodiments, provided herein are one or more sgRNA(s) selected from sequences represented by SEQ ID Nos. 1-4, 9 and 10 for use in detecting SARS-CoV-2 in a sample; preferably an Omicron variant of SARS-CoV-2. In some embodiments, envisaged herein are one or more sgRNA(s) selected from sequences represented by SEQ ID Nos. 1-4, 9 and 10 for use in detecting subvariants BA.1, BA.2 and/or BA.3 of the Omicron variant of SARS-CoV-2 in a sample. As per the requirements of the CRISPR technology, the aforesaid sgRNA(s) are used in conjunction with a suitable Cas nuclease, such as but not limited to Cas12a or its orthologs as defined earlier in the present disclosure to facilitate detection of the Omicron variant of SARS- CoV-2. The ‘detection’ as described above, is facilitated by exploiting the cleavage efficiency of the CRISPR-Cas system, wherein the guides are designed such that they specifically recognize sequences comprising the mutations of interest in the S and/or N proteins of SARS-CoV-2. Thus, the sgRNA sequences are designed such that they can distinguish targets that vary by one or more nucleotide(s) vis-à-vis the non-omicron sequence at one or more positions. Accordingly, in some embodiments, CRISPR mediated cleavage is considered as a confirmative event for the presence of the target (Omicron or non-Omicron) in a sample. In some embodiments, depending on the specific Cas nuclease employed, the assay may utilize the specific or non-specific cleaving activity of the Cas nuclease to render a detectable signal. Method of detection In line with the above, further provided by the present disclosure is a method of detecting SARS- CoV-2 in a sample, comprising contacting the sample with one or more of the CRISPR sgRNA(s) defined above, in presence of a Cas nuclease, a reporter system for the Cas nuclease activity and optionally, buffer and/or water. The Cas nuclease may be selected from a group comprising commonly used Cas nucleases in this domain, as defined above. To enable the aforesaid detection, the reporter system for confirming presence or absence of the mutations characteristic of the Omicron variant of SARS-CoV-2 in the sample is activated upon target recognition by the CRISPR sgRNA, by action of the Cas nuclease. Accordingly, in some embodiments, the Cas nuclease that is part of the CRISPR-Cas system performs a dual role of facilitating cleavage at the recognition site, as well as activating the reporter, for the confirmation of presence of the target of interest. Said activity of the Cas nuclease stems from the choice of Cas nuclease employed. In some embodiments, the Cas nuclease is Cas12a or its orthologs, which possesses a trans-cleavage activity. Thus, in some embodiments, the method of detecting SARS-CoV-2 in a sample, comprises contacting the sample with one or more of the CRISPR sgRNA(s) defined above, in presence of Cas12a nuclease, a reporter system for Cas12a trans-cleavage activity and optionally, buffer and/or water. In some embodiments, the reporter system produces a detectable signal to indicate a detection event. In some embodiments, reporters employable in the above method may provide signals detectable by fluorescence, luminescence, radioactivity, colorimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. In some embodiments, the reporter system is selected from but not limited to radioactive labels, enzymes, or chemiluminescent or bioluminescent or fluorescent moieties. In order to effect detection of the analyte/guide hybrid, reporters may be incorporated into reporter reagents comprising a reporter molecule linked to an immuno-reactive or affinity reactive member of a binding pair. In some embodiments, the reporter system comprises fluorescent reporter molecules such as but not limited to SYT09, fluorescein, rhodamine, rhodoamine600, R-phycoerythrin, and Texas Red. In some embodiments, the reporter system comprises a reporter pair designed to be present at the termini of a nucleic acid strand the wherein upon recognition of the target sequence by the guide sequence of the CRISPR-Cas system and hence activation of activity of the Cas nuclease, members of the reporter pair are designed to be separated by cleavage of the nucleic acid by the Cas nuclease, resulting in signal modification that can then be detected. In some embodiments, the reporter system is in the format of F-(N)n-Q or Q-(N)n-F; wherein F is a fluorescent reporter molecule such as but not limited to SYT09, fluorescein, rhodamine, rhodoamine600, R-phycoerythrin, and Texas Red; wherein N is selected from A, G, T, C, rA, rG, rT and rC; wherein Q is a quencher such as but not limited to black hole quencher (BHQ), Iowa black and 4-(dimethylaminoazo)benzene-4-carboxylic acid (DABCYL) and wherein n ranges from about 6 to about 15. Such a reporter system has been referred to as ssDNA_FQ reporter in the examples section. In some embodiments, the sample/nuclease reaction in the presence of the aforesaid reporter molecule forms a product which results in a detectable signal, typically a change in color. In many cases, chromogenic substances are an additional requirement for the color reaction. Accordingly, in some embodiments, when the method employs enzymes/nucleases as reporters, in order for production of a detectable signal, chromogenic substance(s) may be relied upon. Some typical enzyme/chromogen pairs include, but are not limited to; β-galactosidase with chloro-phenol red β- δ-galactopyranoside (CPRG), potassium ferrocyanide or potassium ferricyanide; horse-radish peroxidase with 3,3′ diaminobenzidine (DAB); glucose oxidase with nitro-blue tetrazolium chloride (NBT), alkaline phosphatase with para-nitrophenyl phosphate (PNPP), or 5-bromo-4- chloro-3-indolylphosphate-4-toluidine (BCIP)/NBT. In exemplary embodiments, the Cas nuclease of the CRISPR-Cas system employed in the present disclosure is Cas12a. In a non-limiting embodiment, the reporter system employed in the method of the present disclosure leverages the trans-cleavage activity of Cas12a. Without intending to be limited by theory, Cas12a enzyme binds to an sgRNA to make Cas12a:sgRNA complex. The Cas12a: guide RNA complex, in the presence of target DNA, makes a trimeric nucleoprotein complex, Cas12a: sgRNA:target DNA. This trimeric nucleoprotein complex possesses a non- specific endonuclease activity and it cleaves ssDNA irrespective of its sequence. If the termini of an ssDNA are labeled with a fluorophore and quencher pair, its cleavage by Cas12a:sgRNA:target DNA complex will lead to an increase in fluorescence. Such an ssDNA can therefore be used as a reporter to detect the presence of Cas12a: sgRNA:target DNA or indirectly the target DNA. Accordingly, in some embodiments, reagents employed in the above-described method of the present disclosure may comprise Cas12a enzyme, one or more sgRNA(s) selected from a group comprising sequences represented by SEQ ID Nos.1-4, and a reporter such as an ssDNA labeled with a fluorophore and quencher pair. If the sample subjected to detection contains the target DNA; the Cas12a:guide RNA:target DNA forms and cleaves the ssDNA resulting in an increase in fluorescence signal. Said embodiments of the present disclosure therefore make use of the trans- cleavage mechanism of the Cas12a nuclease. In some embodiments, the method employs one or more additional sgRNAs against a target in the SARS-CoV-2 genome different from that targeted by any of the SEQ ID No. 1, SEQ ID No. 2, SEQ ID No.3 and SEQ ID No.4. Without intending to be limited by theory, the additional sgRNA gives a positive signal for both Omicron and non-Omicron variants of the SARS-CoV-2, confirming the sample to be SARS-CoV-2 positive and avoiding false negative results. In a non-limiting embodiment, the additional sgRNAs targets ORF1ab gene in SARS-CoV-2. In a non-limiting embodiment, the additional sgRNAs targeting the ORF1ab gene in SARS-CoV- 2 have sequences represented by SEQ ID No.9 and SEQ ID No.10. Thus, in some embodiments, reagents employed in the above-described method of the present disclosure may comprise Cas12a enzyme, one or more sgRNA(s) selected from a group comprising sequences represented by SEQ ID Nos.1-4, 9 and 10, and a reporter such as an ssDNA labeled with a fluorophore and quencher pair. Accordingly, the method of detecting SARS-CoV-2 in a sample comprises contacting the sample with one or more of the CRISPR sgRNA(s) represented by SEQ ID Nos.1-4, 9 and 10, in presence of a Cas nuclease, a reporter system for the Cas nuclease activity and optionally, buffer and/or water; wherein preferably the Cas nuclease is Cas12a. In some embodiments, the method may be characterized by reactions employing each of the separate sgRNAs occurring simultaneously or sequentially. As per the known mechanism of the Cas nuclease, a protospacer adjacent motif (PAM) is required for a Cas nuclease to perform cleavage and is generally found 3-4 nucleotides downstream from the cut site. In some embodiments, when the target locus does not naturally contain a PAM sequence, the target locus is converted to a Cas susceptible site by artificially introducing a PAM sequence. In some embodiments, the PAM sequence is artificially introduced by using primer(s) designed to comprise the PAM sequence, for amplification of the target loci; wherein the amplification of the target loci takes place within the device or prior to application to the device. Sequence(s) amplified using said primers therefore comprise the PAM sequence. The above-described method, in a non-limiting embodiment, is performed on a biological sample or an environmental sample, wherein the said sample comprises nucleic acid potentially containing the target of interest. Such samples employable in the method of the present disclosure may be individual samples or mixed samples. In some embodiments, the said sample is an unprocessed sample or a pre-processed sample. An unprocessed sample is any biological sample which can act as source of genetic material such as but not limited to blood, urine, feces, sperm, saliva, tissue biopsy and intraoral mucosa, or an environmental sample such as but not limited to sewage, food sample, surface swab and milk that presents a mixed matrix. Examples of pre-processed samples include but are not limited to nucleic acids isolated and/or amplified from biological or environmental samples. The rate of the CRISPR-Cas mediated cleavage is dependent on the target template concentrations. At higher concentrations the rates of cleavage are faster, and to capture the differences in fast rates of reaction, a higher time resolution in data points is required. In preferred embodiments, nucleic acids from the sample are amplified at the regions of the target loci, and subsequently subjected to the step of detection. In some embodiments, the amplification is achieved by methods such as but not limited to Polymerase Chain Reaction (PCR), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), ligase chain reaction (LCR), Nucleic Acid Sequence Based Amplification (NASBA), transcription-associated amplification (TAA), Cold PCR, and Non-Enzymatic Amplification Technology (NEAT). In an exemplary embodiment, the amplification is achieved by Polymerase Chain Reaction (PCR). In a non-limiting embodiment, the primers employed for amplification are targeted towards the loci of interest i.e. the target of SEQ ID Nos.1 and 3 or SEQ ID Nos.2 and 4. Accordingly, further provided herein are primers that may be employed for facilitating amplification of the sample at the loci of interest. The primer(s) are designed as per the sequence at the target loci or template. The primers are designed such that minimal amount of non-specific amplification is detected and further, such that amplification of multiple amplicons is facilitated through a minimum number of reactions. In some embodiments, as explained above, the primers are designed to comprise the PAM sequence to yield Cas susceptible amplified nucleic acids. In some embodiments, the primer(s) have sequences represented by SEQ ID Nos.11-18. Therefore, in some embodiments, the method of the present disclosure comprises contacting the sample with one or more of the CRISPR sgRNA(s) defined above, in presence of a Cas12a nuclease, a reporter system for the Cas12a nuclease activity and optionally, buffer and/or water; wherein the sample is an amplified sample. In some embodiments, the amplification is performed prior to or simultaneously with the detection. In some embodiments, the amplification is optionally preceded by nucleic acid isolation. Accordingly, in some embodiments, the method of the present disclosure comprises optionally isolating nucleic acid and/or subjecting nucleic acid from the sample to amplification; contacting the isolated and/or amplified nucleic acid with one or more of the CRISPR sgRNA(s) defined above, in presence of a Cas12a nuclease, a reporter system for the Cas12a nuclease activity and optionally, buffer and/or water. In some embodiments, the isolation and/or amplification is performed simultaneously along with the detection. In some embodiments, the method of the present disclosure comprises contacting the isolated and/or amplified nucleic acid with one or more of the CRISPR sgRNA(s) defined above, in presence of primers and reagents to facilitate isolation or nucleic acid and/or their amplification, a Cas12a nuclease, a reporter system for the Cas12a nuclease activity and optionally, buffer and/or water. In some embodiments, the method of the present disclosure comprises - Subjecting nucleic acid in the sample to amplification at the target loci to obtain an amplified sample, - Contacting the amplified sample with one or more of the CRISPR sgRNA(s) defined above, in presence of a Cas12a nuclease, a reporter system for the Cas12a nuclease activity and optionally, buffer and/or water. In some embodiments, the method of the present disclosure comprises - Isolating nucleic acid from the sample, - Subjecting isolated nucleic acid to amplification at the target loci to obtain an amplified sample, - Contacting the amplified sample with one or more of the CRISPR sgRNA(s) defined above, in presence of a Cas12a nuclease, a reporter system for the Cas12a nuclease activity and optionally, buffer and/or water. In some embodiments, the method of the present disclosure comprises - Isolating nucleic acid from the sample, - Subjecting isolated nucleic acid to amplification using primer(s) selected from a group comprising SEQ ID Nos.11-18 at the target loci to obtain an amplified sample, - Contacting the amplified sample with one or more of the CRISPR sgRNA(s) selected from a group comprising SEQ ID Nos.1-4, 9 and 10, in presence of a Cas12a nuclease, a reporter system for the Cas12a nuclease activity and optionally, buffer and/or water. In exemplary embodiments, the amplification of sample (isolated nucleic acid or the sample as is, comprising nucleic acids) is performed at conditions that facilitate the amplification of nucleic acids, said conditions being well known to a person skilled in the art. In some embodiments, the buffer comprises Bovine Serum Albumin (BSA) in combination with other standard buffer components such as but not limited to salt(s) that act as buffering agents that resist one or more of osmotic shock, pH change and change in ionic strength. In other words, the buffer employed in the method of the present disclosure, in some embodiments, is a BSA containing buffer. In some embodiments, the buffer as referred to above comprises components such as but not limited to Tris-HCl, MgCl2, NaCl, Tris-Acetate, Magnesium Acetate, Potassium Acetate in combination with BSA. In some embodiments, the buffer comprises about 5 mM to about 15 mM of Tris-HCl, about 5 mM to about 15 mM of MgCl2, about 45 mM to about 55 mM of NaCl, and about 90 µg/mL to about 110 µg/mL of BSA. In some embodiments, the buffer comprises about 10 mM of Tris-HCl, about 10 mM of MgCl2, about 50 mM of NaCl and about 100 µg/mL of BSA. In some embodiments, the buffer comprises about 15mM to about 25 mM_of Tris-acetate, about 45 mM to about 55 mM of Potassium Acetate, about 5mM to about 10 mMof Magnesium Acetate and about 90 µg/mL to about 110 µg/mL of BSA. In some embodiments, pH of the buffer ranges from about 7 to about 7.9. In some embodiments, the buffer may comprise additional reagents to optimize the detection reaction. In some embodiments, the aforesaid method has analytical limit of detection ranging from about 100 copies to about 500 copies. In a non-limiting embodiment, in case of a clinical sample, the aforesaid method has analytical limit of detection of about 100 copies. In another non-limiting embodiment, in case of an environmental sample such as a sewage sample, the aforesaid method has analytical limit of detection ranging from about 200 copies to about 500 copies. In some embodiments, the aforesaid method has specificity ranging from about 80% to about 100%. In a non-limiting embodiment, in case of a clinical sample, the aforesaid method has specificity of about 100%. In some embodiments, the aforesaid method has sensitivity ranging from about 80% to about 100%. In a non-limiting embodiment, in the case of a clinical sample, the aforesaid method has sensitivity ranging from about 94% to about 100%. In some embodiments, the aforesaid method is implemented as an in-vitro assay; wherein the in- vitro assay is performed in a multi-well plate, multi-strip tube, individual tube(s) or similar platform; wherein each well comprises a different CRISPR sgRNA along with a Cas nuclease, a reporter pair and optionally, buffer and/or water. In some embodiments, the aforesaid method is implemented as an in-vitro assay; wherein the in- vitro assay is performed in a multi-well plate, multi-strip tube, individual tube(s) or similar platform; wherein each well comprises a different CRISPR sgRNA along with a Cas12a nuclease, ssDNA labelled with a fluorophore and quencher pair and optionally, buffer and/or water. In order to facilitate the above method, the present disclosure further provides an assay device that provides a detectable signal or readout confirming the presence or absence of the target of interest detectable by one or more of the sgRNAs of the present disclosure. Assay device In order to facilitate the detection of SARS-CoV-2 and/or the Omicron variant or sub-variant of SARS-CoV-2, the present disclosure further provides an assay device that allows CRISPR mediated detection of signature mutations in the Omicron variant of SARS-CoV-2 infection, characterized by incorporation of specifically designed CRISPR sgRNA sequences as described above. In some embodiments, the assay device is designed to comprise a patterned surface suitable for immobilization of molecules in an ordered pattern. In some embodiments a patterned surface refers to an arrangement of different regions in or on an exposed layer of a solid support. In some embodiments, the solid support comprises an array of wells or depressions on a surface, or a lateral flow strip made of paper or any membrane. The composition and geometry of the solid support can vary with its use. In some embodiments, the solid support is a planar structure such as a slide, chip, flow strip, microchip and/or array. In some embodiments, the assay device is designed in the form of cartridges or reaction vessels, or series or arrays thereof. In some embodiments, the assay is facilitated on beads or in solution form. Accordingly, in some embodiments, the assay device is composed of beads and means to hold the same or containers comprising the assay reagents in solution, or series or arrays of said containers or set of beads. In some embodiments, the assay device is made of any material that allows for the immobilization of a polypeptide, a polynucleotide or a protein-nucleic acid complex. In some embodiments, the assay device is made of conductive or non-conductive material. In some embodiments, the assay device is made of non-conductive material and coated with conductive substances to confer to the platform electrical and/or thermal conductivity. In some embodiments, the assay device is made of made of material such as but not limited to glass, modified functionalized glass, plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silicon and modified silicon, carbon, metals, inorganic glasses, plastics, optical fiber bundles, and or any other polymers. The ‘detection’ in the device of the present disclosure is facilitated by exploiting the cleavage efficiency of the CRISPR-Cas system comprising the sgRNAs as defined above. In some embodiments, the said CRISPR-Cas system may be immobilized on to the solid substrate or is suspended in liquid medium of the assay device. In some embodiments, the CRISPR-Cas system is directed towards target loci in samples that may be immobilized in separate wells or depressions on the surface of the assay device or contained on different sets of beads or in different solutions. In some embodiments, the CRISPR-Cas system may be incorporated into the device in any of the above forms as free nucleotides (sgRNA) and peptides (Cas nuclease). In some embodiments, the CRISPR-Cas system may be present in the device in encapsulated form, wherein said encapsulation may be in vehicles such as but not limited to liposomes or hydrogels. In some embodiments, the assay device is designed such that nucleic acid isolation and amplification occurs within the assay device. Alternatively, in some embodiments, the sample is partially processed so as to isolate the nucleic acids, such that said nucleic acids are applied to the assay device and the amplification at target loci occurs within the assay device. Accordingly, in some embodiments, the assay device may further comprise reagents to facilitate the isolation of nucleic acids from the unprocessed sample and/or amplification at target loci. Said reagents that facilitate the isolation of nucleic acids from the unprocessed sample and/or amplification at target loci include but are not limited to enzyme(s), dNTPs, buffer(s) and primer(s). In a non-limiting embodiment, the enzyme for facilitating amplification is a polymerase enzyme. With regard to the primer(s), the primer(s) include those as described under the embodiments pertaining to the method and are not repeated herein for reasons for brevity. In some events, the assay-based detection may be performed in duplicates for validation of results. In an embodiment, the assay device may optionally comprise means for control detection event(s). In some embodiments, a positive control detection event may employ a sample known to be an Omicron positive sample. In some embodiments, the negative control detection event may not employ a sample. In some embodiments, the assay device is designed such that each detection event – directed towards the N protein of SARS-CoV-2, the S protein of SARS-CoV-2, an additional target in the SARS-CoV-2 genome different from the target in the N and S proteins of SARS-CoV-2 and an optional positive and/or negative control - occurs in separate wells or depressions on the surface of the assay device, or on separate set of beads or separate solutions within the assay device. In some embodiments, the assay device is designed such that each detection event – directed towards the N protein of SARS-CoV-2, the S protein of SARS-CoV-2, an additional target in the SARS-CoV-2 genome different from the target in the N and S proteins of SARS-CoV-2 and an optional positive and/or negative control - occurs in the same reaction well or depression on the surface of the assay device, or on the same set of beads or same solution within the assay device. In some embodiments, reagents in the assay device comprise sgRNA selected from a group comprising sequences represented by SEQ ID Nos.1-4, 9 and 10, Cas nuclease and a reporter such as an ssDNA labeled with a fluorophore and quencher pair. In some embodiments, the Cas nuclease is Cas12a and if the sample to be detected contains the target DNA; the Cas12a:guide RNA:target complex gets formed and cleaves the ssDNA resulting in an increase in fluorescence signal. In some embodiments, reagents in the assay device comprise reagents that facilitate the isolation of nucleic acids from the unprocessed sample and/or amplification at target loci such as but are not limited to polymerase enzyme(s), dNTPs, buffer(s) and primer(s), sgRNA selected from a group comprising sequences represented by SEQ ID Nos.1-4 , 9 and 10, Cas12a nuclease and a reporter such as an ssDNA labeled with a fluorophore and quencher pair. In some embodiments, the reactions in the assay device that enable genotyping of the above defined SNPs are optimized by controlling parameters such as temperature, pH, duration of assay, template concentration and salt composition. In some embodiments, the assay device disclosed herein is prepared in freeze-dried format for convenient distribution. However, when put to application, the assay device performs the assay at ambient temperature, preferably ranging from about 25°C to about 37°C. In some embodiments, the assay device optionally comprises attachments such as electrodes for introducing electric field or electrically or battery-controlled means of regulating temperature to optimize the performance of the assay device when put to application at the point of care. In some embodiments, the assay device comprises a reaction buffer that facilitates action of the Cas nuclease. In some embodiments, the reaction buffer comprises components such as but not limited to Tris-HCl, MgCl2, NaCl, and BSA. In some embodiments, the reaction buffer comprises about 5 mM to about 15 mM of Tris-HCl, about 5 mM to about 15 mM of MgCl2, about 45 mM to about 55 mM of NaCl, and about 90 µg/mL to about 110mg/mL of BSA. In an exemplary embodiment, the reaction buffer comprises about 10 nM of Tris-HCl, about 10 mM of MgCl2, about 50 mM of NaCl and about 100 µg/mL of BSA. In some embodiments, pH of the reaction buffer ranges from about 7 to about 7.9. In some embodiments, the reaction buffer may comprise additional reagents to optimize the detection reaction in the assay device. In some embodiments, the reaction buffer is already present in the wells, depressions or containers of the assay device before the sample is applied or is added post application of the sample to the assay device. In some embodiments, the reaction buffer is the same buffer employed for amplification of the target loci. Accordingly, in some embodiments, the reaction buffer is introduced into the assay device along with the sample as part of an amplification mix comprising the amplified target loci. In some embodiments, the sample is a freshly collected sample or a sample previously collected at another location. In some embodiments, the sample is an unprocessed sample or a pre-processed sample as described in the previous embodiments relating to the method. Said embodiments are not re-iterated herein for reasons of brevity. The present disclosure further provides a method of using the aforesaid assay device for the detection of SARS-CoV-2 and/or the Omicron variant of SARS-CoV-2 in a sample comprising – applying the sample to the assay device described above to obtain a detectable signal. Application of the assay device In some embodiments, the present disclosure provides a method of using the aforesaid assay device for the detection of SARS-CoV-2 and/or the Omicron variant of SARS-CoV-2 in a sample comprising– extracting nucleic acid from the sample; applying the extracted nucleic acid to the assay device described above to obtain a detectable signal. In some embodiments, the present disclosure provides a method of using the aforesaid assay device for the detection of SARS-CoV-2 and/or the Omicron variant of SARS-CoV-2 in a sample comprising– extracting nucleic acid from the sample; amplifying target loci in the extracted nucleic acid; applying the amplified target loci to the assay device described above to obtain a detectable signal. In some embodiments, the present disclosure provides a method of using the aforesaid assay device for the detection of SARS-CoV-2 and/or the Omicron variant of SARS-CoV-2 in a sample comprising– extracting nucleic acid from the sample; amplifying target loci in the extracted nucleic acid using primer(s) selected from a group of sequences represented by SEQ ID Nos.11-18; and applying the amplified target loci to the assay device described above comprising CRISPR-Cas system having sgRNA(s) selected from a group of sequences represented by SEQ ID Nos.1-4 and 9-10; to obtain a detectable signal. In some embodiments, the extraction of nucleic acid and amplification of target loci may be performed by any method known in the art. In some embodiments, the extraction and amplification of target loci may be performed before application of the sample to the assay device or within the assay device itself, or in an extension thereof. The detectable signal in the aforesaid method is observed by virtue of the CRISPR-Cas system recognizing and cleaving the target (risk allele and/or alternative allele). In some embodiments, said recognition of the target allele is accompanied by simultaneous activation of the reporter system by the Cas nuclease, yielding a detectable (preferably quantifiable) signal. In exemplary embodiments, the detectable signal is yielded by a fluorometric or a colorimetric reaction. In some embodiments, the detectable signal is observable by the naked eye. Such an observation provides an indication of the presence or absence of the risk allele and/or the alternative allele. For quantification, in some embodiments, the signal is measured using a spectrophotometer, a colorimeter, a fluorometer or a luminometer depending on the reporter system employed. In some embodiments, the method envisaged in each of the above embodiments is an in-vitro method. The present disclosure therefore provides an in-vitro method of using the aforesaid assay device for the detection of SARS-CoV-2 and/or the Omicron variant of SARS-CoV-2 in a sample comprising– applying the sample to the assay device described above to obtain a detectable signal. In some embodiments, the present disclosure provides an in-vitro method of using the aforesaid assay device for the detection of SARS-CoV-2 and/or the Omicron variant of SARS-CoV-2 in a sample, wherein said method comprises – extracting nucleic acid from the sample; and applying the extracted nucleic acid to the assay device described above to obtain a detectable signal. In some embodiments, the present disclosure provides an in-vitro method of using the aforesaid assay device for the detection of SARS-CoV-2 and/or the Omicron variant of SARS-CoV-2 in a sample, wherein said method comprises – extracting nucleic acid from the sample; amplifying target loci in the extracted nucleic acid using primer(s) selected from a group of sequences represented by SEQ ID Nos.11-18; and applying the amplified target loci to the assay device described above comprising CRISPR-Cas system having sgRNA(s) selected from a group of sequences represented by SEQ ID Nos.1-4 and 9-10; to obtain detectable signal. In some embodiments, the present disclosure provides an in-vitro method of using the aforesaid assay device for the detection of SARS-CoV-2 and/or the Omicron variant of SARS-CoV-2 in a sample, wherein said method comprises – extracting nucleic acid from the sample; amplifying target loci in the extracted nucleic acid to obtain an amplification mix comprising the amplified target loci; and applying the amplification mix nucleic acid to the assay device described above to obtain a detectable signal. In some embodiments, the present disclosure provides an in-vitro method of using the aforesaid assay device for the detection of SARS-CoV-2 and/or the Omicron variant of SARS-CoV-2 in a sample, wherein said method comprises – extracting nucleic acid from the sample; amplifying target loci in the extracted nucleic acid to obtain an amplification mix comprising the amplified nucleic acid using primer(s) selected from a group of sequences represented by SEQ ID Nos.11-18; applying the amplification mix nucleic acid to the assay device described above comprising CRISPR-Cas system having sgRNA(s) selected from a group of sequences represented by SEQ ID Nos.1-4 and 9-10, to obtain a detectable signal. In some embodiments, the aforesaid method is performed at ambient temperature. In a non-limiting embodiment, the aforesaid method is performed at a temperature ranging from about 25°C to about 37°C. In some embodiments, the aforesaid method is performed at the point of sample collection (point of care) or in a separate setting from the point of sample collection. In exemplary embodiments, the aforesaid method is performed at the point of care. In some embodiments, the aforesaid method provides a detectable signal or readout in about 10 minutes to about 120 minutes after application of the amplified nucleic acid or the amplification mix. In some embodiments, the detectable signal is observed as a qualitative reading merely confirming presence or absence of the target in a sample. In some embodiments, the detectable signal is measured using a spectrophotometer, a colorimeter, a fluorometer or a luminometer depending on the reporter system employed. Accordingly, the assay of the present disclosure provides scope for point-of-care detection of SARS-CoV-2 in a sample and further confirms the presence or absence of the Omicron variant of SARS-CoV-2 in the sample. In some embodiments, the aforesaid method has analytical limit of detection ranging from about 100 copies to about 500 copies. In a non-limiting embodiment, in case of a clinical sample, the aforesaid method has analytical limit of detection of about 100 copies. In another non-limiting embodiment, in case of a sewage sample, the aforesaid method has analytical limit of detection ranging from about 200 copies to about 500 copies. In some embodiments, the aforesaid method has specificity ranging from about 80% to about 100%. In a non-limiting embodiment, in case of a clinical sample, the aforesaid method has specificity of about 100%. In some embodiments, the aforesaid method has sensitivity ranging from about 80% to about 100%. In a non-limiting embodiment, in case of a clinical sample, the aforesaid method has sensitivity ranging from about 94% to about 100%. Further envisaged in the present disclosure are automated forms of the method(s) and the assay as described above, wherein said automation, in line with the underlying concept and design of the present disclosure, would be well within the purview of knowledge of a skilled artisan and would require no inventive effort as such. Data interpretation – Omicron vs. Non-Omicron The below method of calculation or data interpretation provides mere exemplification of one of the possible ways of interpretation and is only intended to be illustrative in scope and not restrictive. A person skilled in the art may rely on other formulae or steps to interpret the signals derivable from the method or assay device as described above to arrive at the same conclusion with respect to the sample subjected to detection using the sequence(s) and method(s) of the present disclosure. As mentioned in some embodiments, the methods of the present disclosure may be used for detecting and confirming the presence of Omicron variant of SARS-CoV-2 in a sample. In some embodiments, variant prediction may be performed using the S gene targeting sgRNA i.e. SEQ ID No.1 and 3 or the N gene targeting sgRNA i.e. SEQ ID No.2 and 4, independently. In situations where variant predictions cannot be made using either of the S or N gene, the assay may be repeated. If prediction can be made only with one gene out of S and N, the corresponding prediction may be reported as the final result for the sample. In some embodiments, if the predictions can be made using both S and N genes, and they do not match, the sample may be labeled ambiguous with suspected mixed sample or contamination. In a non-limiting embodiment, the predictions/data interpretation using N gene and S gene may be made as described below: Threshold signal estimation Every time a trans-cleavage assay is performed, negative controls are included for each guide in the used for the trans-cleavage. The negative control has all the reagents added except the sample. Signals from negative controls may be used to estimate the noise or fluctuation in the signal. The noise can be used to estimate the threshold signal. The threshold signal is the value that can be considered as higher than background signal. The calculation for noise and threshold signal are described below. The signal from the negative controls of all the guide RNAs are pooled into one set and called as “pooled negative controls”. The median and interquartile range of the “pooled negative controls” is calculated using standard formulas. In some embodiments, first, outliers are removed from the “pooled negative controls” (which could arise from random contamination). Upper limit of outlier = median negative control signal + 1.5*Interquartile range (IQR) All the negative control points with signals higher than the Upper limit of outlier as defined above are removed. In some embodiments, in case of a situation where more than 20 % of the data points are higher that upper limit of outlier, the entire assay may be discarded and repeated with fresh reagents. After removal of these points standard deviation is calculated of the remaining “pooled negative data set” using standard formulas. After removing the outliers in the noise, threshold signal, and reverse transcription negative control (RT-NTC) cut off may be calculated as described below. Noise = 3* Standard deviation of signal of pooled detection negative controls after outlier removal Threshold signal (Thresh_signal)= 1.2*Noise The number ‘1.2’ in the present disclosure was estimated from the data that was acquired from the validation on clinical samples. This value is dependent on the sample type and may be subject to change. RT-NTC cut off = median of pooled negative controls + 1.5*Thresh_signal Unreliable data removal In order to interpret results of the assay for a given sample, first the reliability of the signals obtained after trans-cleavage with all six guides is evaluated. The reliability of the signals is estimated by estimating the variations in technical duplicates of the trans-cleavage assay for each guide for each sample. And any data points which show large variations in duplicates are considered unreliable and hence removed before the interpretation of the assay. In some embodiments, in order to remove the data points with large variation in signal in technical duplicates, percentage relative standard deviation (%RSD) for technical duplicates may be calculated as described below. %RSD for one guide for a given sample = (Standard deviation of duplicate*100)/(average of duplicate) All the data points with %RSD more than about 20% may be removed from the analysis. If the RT-NTC of a given guide is above RT-NTC cutoff or its %RSD is greater than 20% all the data points for that guide for all the samples may be removed. In case of a situation where after removing the unreliable data less than three SARS-CoV-2 guides are left for analysis, the assay for that sample may be repeated. SARS-CoV-2 positive versus negative prediction In some embodiments, in

signal for a given guide is positive or negative in a sample, the background subtracted signal (Back sub signal) is estimated. (Back sub signal is the signal obtained by subtracting the signal for RT-NTC control. RT-NTC control is where in place of sample, water is added as an input at the step of amplification. Back sub signal = average signal of a given guide for a sample - average signal of the same guide for RT-NTC If Back sub signal>Thresh signal label it is labeled as positive signal, else it is labeled as negative signal. The number SARS-CoV-2 gene per sample with positive signal is calculated. Taking the S gene as an example, the S gene has two guides: SRef and SOm. S gene may be counted as one gene positive if only one or both the S gene guides have positive signal. Similarly, the N gene has two guides: NRef and NOm. The N gene may be counted as one gene positive if only one or both the N gene guides have positive signal. If for a sample no SARS-CoV-2 genes are positive, that sample is labeled as “SARS-CoV-2 negative”. If for a sample 1 SARS-CoV-2 gene (out of ORF, S, and N) has positive signal, it may be reported as “repeat to confirm it is negative”. If for a sample 2 or 3 SARS-CoV-2 gene (out of ORF, S, and N) has a positive signal, it may be reported that the sample is “SARS-CoV-2 positive”. Variant prediction using S gene guides If both

variant prediction is not made with S gene. In some embodiments, if signals for both the guides for S gene, i.e. SOm and SRef, are available, the SOm/SRef may be calculated as described below; SOm/SRef = Back sub signal of SOm/ Back sub signal of SRef If SOm/SRef >0.5, the sample is labeled as “Omicron”, else it is labelled as “Not-Omicron”. The value 0.5 is obtained from the validation data set. This depends on the sample type used for the assay and therefore, may be subject to change based on the sample employed. Variant prediction using N gene guides If both N^Om and N^Ref signals are not available, variant prediction is not made using the N gene. If signals for both the guides for N gene, i.e. NOm and NRef, are available irrespective, the NOm/NRef is calculated as described below; NOm/NRef= Back_sub_signal of NOm /Back _sub signal of NRef If, NOm/NRef >1, the sample is labelled as “Omicron”, else it is labelled as “Not-Omicron” Final variant prediction may be done as described below: 1. If only one of the N or S gene could be used for variant prediction, the results corresponding to the variant prediction of that gene is reported. 2. If both N and S gene could be used for prediction and the predictions of both are found to be the same, the result observed for both is reported. 3. If N and S gene variant prediction do not match, the result reported is, “Possibility of mixed sample or contamination. The assay may be repeated with freshly collected samples to overrule the possibility of contamination.” 4. If neither N nor S gene could be used for variant prediction, the assay may be repeated because the data may not be sufficient for variant prediction. Without intending to be limited by theory, in some embodiments, the method of detection and/or the method of interpretation of the results may be automated. Kit The present disclosure further provides a kit comprising the assay device as defined above or the sgRNA sequences as defined above, optionally along with an instruction manual. In some embodiments, the kit further comprises one or more component(s) selected from a group comprising a multi-well plate, multi-strip tube or individual tube(s), Cas enzyme, reaction buffer(s), means for sample procurement, means for application of the sample to the assay device, primer(s), dNTPs, polymerase enzyme(s) and reporter system(s) or any combination thereof, optionally along with an instruction manual. In some embodiments, the present disclosure provides a kit comprising the CRISPR sgRNA(s) represented by SEQ IID nos.1-4; Cas12a nuclease or orthologs thereof; reporter system for trans cleavage activity of Cas12a nuclease or orthologs thereof; and optionally, water, buffer, additional sgRNA(s) against a target in the SARS-CoV-2 genome different from that targeted by any of the SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, the primers as defined in claim 14, reagents for amplification, a multi-well plate or multi-strip tube or individual tube(s), and an instruction manual. In some embodiments, the kit comprises one or more sgRNAs selected from a group comprising sequences represented by SEQ ID Nos.1-4, 9 and 10 and one or more component(s) selected from a group comprising a multi-well plate, multi-strip tube or individual tube(s), Cas12a enzyme, reaction buffer(s), means for sample procurement, means for application of the sample to the assay device, primer(s) selected from a group comprising sequences represented by SEQ ID Nos.11-18, dNTPs, polymerase enzyme(s) and reporter system(s) or any combination thereof, optionally along with an instruction manual. The reaction buffer(s), reporter system(s) and polymerase enzyme(s) have been described in detail in the earlier embodiments of the present disclosure and are not repeated for reasons of brevity. In a non-limiting embodiment, examples of the means for sample procurement include but are not limited to syringe, nasal swab, throat swab and sample container. In a non-limiting embodiment, examples of the means for sample application include but are not limited to syringe, dropper and pipette. Each of said components are defined in earlier embodiments in the context of the sgRNA, the method or the assay device. In some embodiments, the sgRNAs are immobilized on the multi-well plate, multi-strip tube or individual tube(s) and the multi-well plate, multi-strip tube or individual tube(s) comprising the immobilized sgRNAs constitutes an assay device. In some embodiments, instead of a pre-prepared assay device, the above described kit of the present disclosure may comprise one or more of the sgRNAs of the present disclosure along with the a patterned substrate, set of beads or reaction tubes, along components selected from a group comprising reaction buffer(s), Cas12a nuclease, means to immobilize the CRISPR-Cas system, means to procure a sample, means to apply the sample to the assay device, primer(s), dNTPs, polymerase enzyme(s), reporter system(s) or any combination thereof, optionally along with an instruction manual. The subject matter of embodiments of the present invention is described here with specificity to meet statutory requirements, but this description is not necessarily intended to limit the scope of the disclosure. The disclosed subject matter may be embodied in other ways, may include different elements or steps, and may be used in conjunction with other existing or future technologies. This description should not be interpreted as implying any particular order or arrangement among or between various steps or elements except when the order of individual steps or arrangement of elements is explicitly described. It is to be understood that the foregoing descriptive matter is illustrative of the disclosure and not a limitation. While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein. Descriptions of well-known/conventional methods/steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above-described embodiments, and in order to illustrate the embodiments of the present disclosure certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein. EXAMPLES: Materials and methods Reagents and Equipment LbaCas12a, Alt-R™ L.b. Cas12a (Cpf1) Ultra, custom guide RNAs Alt-R® L.b. Cas12a crRNA, custom ssDNA_FQ reporter were procured from IDT (Integrated DNA Technologies, USA). DNA oligonucleotides used as primers and synthetic templates were custom synthesized from Sigma (USA), and VNIR Biotechnologies (India). NEBuffer™ 2, and BSA (B9000S) were purchased from NEB (New England BioLabs, USA). The fluorescence measurements were acquired using VarioSkan LUX micro plate reader (Thermo Scientific, USA) in Corning® 96 Well Black Polystyrene Microplates (CLS3603-48EA, Sigma, USA). The fluorescence measurements for OmiCrisp_v2 were acquired in Bio-Rad CFX96 Real-Time PCR machine (Bio-rad, USA). The amplification reactions were performed either in Mastercycler Nexus Thermal Cycler™ (Eppendorf, Germany) or in Applied Biosystems 2720 thermal cycler (Thermo Scientific, USA). SARS-CoV-2 synthetic RNA controls for reference strain (Wuhan-Hu-1, cat. 102024) and Omicron strain BA.1 (Omicron EPI_ISL_6841980, cat.105204) were procured from TwistBiosciences, USA. PCR master mix 2X, K01721, was procured from Thermo Scientific. QIAquick Gel Extraction Kit (cat.28704, Qiagen, Germany) was used for purification of PCR products. Synthetic DNA target preparation Synthetic target DNA corresponding to the region of interest of S gene that harbors Q493R, G496S, and Q498R mutations corresponding to Omicron BA.1 and BA.2 lineages were prepared by the overlap extension of the synthetic oligonucleotides. Briefly, the overlap extension mix was prepared by mixing overlapping oligonucleotides at a concentration of 1 µM in 1X PCR master mix and overlap extension was performed in a thermal cycler with following conditions: Initial heat denaturation at about 95 ℃ for about 5 minutes; 15 cycles of three step amplification that includes first step of heat denaturation at about 90℃ for about 30 seconds , second step of annealing at about 60 ℃ for about 30 seconds, and third step of extension at about 72 ℃ for about 30 seconds; and final extension for about 5 minutes at about 72 ℃. Rest of the synthetic DNA target was prepared by one step RT-PCR amplification from the corresponding synthetic RNA controls, purchased from TwistBiosciences, using a suitable primer pair. About 250 nM of each primer and about 1*10^5 copies of synthetic RNA control were used as input sample for the RT-PCR amplification. The amplification conditions were same as described ahead for multiplexed one step RT-PCR. The products obtained after overlap extension and one step RT-PCR were purified using a purification kit and quantified based on absorbance at about 260 nm in NanoDrop One™ (Thermo Scientific, USA). For converting the concentration into molarity, the molecular weight of DNA was calculated by multiplying the length of the target DNA by average molecular weight of DNA base pair, i.e.660 daltons. Multiplexed one step RT-PCR One step RT PCR was performed using commercially available one step RT PCR mix. Briefly, 4 primer pairs i.e. a total of 8 primers, were added in a single tube to amplify fragment of interest corresponding to ORF1ab, S gene, N gene, and human RNaseP. The concentration of each primer in the reaction mix was about 250 nM. Following thermal cycling conditions were used for the one step RT-PCR: Step 1. primer incubation at about 25 ℃ for about 2 minutes, Step 2. reverse transcription at about 53 ℃ for about 10 minutes, Step 3. enzyme denaturation at about 95 ℃ for about 2 minutes, and Step 4.35-40 cycles of amplification that includes incubation at about 95 ℃ for about 15 seconds for denaturation and combined extension/annealing at about 60 ℃ for about 1 minute. Trans-cleavage assay All the trans-cleavage assays for a given target or sample were carried out using about 25 nM of Cas nuclease LbaCas12a, about 25 nM of the indicated guide RNA in a solution containing about 50 mM NaCl, about 10 mM Tris-HCl, about 10 mM MgCl2, about 50 nM ssDNA_FQ reporter, and about 100 µg/mL BSA (pH 7.9, at about 25 °C) at a temperature of about 37 ℃. For end-point assays, the reactions were stopped after about 1 hour of incubation by adding about 10 µL of stop buffer (about 250 mM EDTA and about 37.5 mM Tris-HCl pH 7 at about 25 ℃) to about 50 µL of the reaction mix. Data was acquired using excitation wavelength of about 485 nm and emission as collected at about 525 nm. For real time assays, all the reactions were initiated by addition of the target samples, and it was ensured that the time difference between the beginning of addition of sample to the first well and the start of the data acquisition was not more than about 2 minutes. EXAMPLE 1: Guide design In order to develop a nucleic acid-based assay for the detection of Omicron variant, mutations that were specific to Omicron variant of SARS-CoV-2 and not associated with any other SARS-CoV- 2 variant were identified. Mutations in the S gene of the Omicron variant that were present at a frequency higher than about 90%, and not present in any of the non-Omicron variants of SARS-CoV-2 at a frequency of higher than about 0.1% were identified, out of which the subset of mutations that were present in all the three variants of Omicron, BA.1, BA.2, and BA.3, of SARS-CoV-2 (reported at the time of research), was shortlisted. The sequences encompassing the shortlisted mutations were examined and the ones that matched the criteria for developing the Cas12a trans-cleavage based assay for variant detection were identified. For each of these mutations that can potentially distinguish Omicron from non-Omicron variant of SARS-CoV-2, a pair of guide RNA was designed. One of the guide RNA was labelled as “Reference-specific guide” and another as another as “Omicron-specific guide”. Trans-cleavage assay done with “Reference-specific guide” was expected to give at least five fold higher signal in the presence of target that does not carry the selected mutation(s) than in the presence of the target that carries the mutation(s), and trans-cleavage assay done with “Omicron-specific guide” was expected to give at least five fold higher signal in presence of target that carries the selected mutation(s) than in the presence of the target that does not carry the mutation(s). Optimization In order to test the selectivity of the designed guide RNA pairs to discriminate Omicron from non- Omicron variants experimentally, the target region, targeted by guide RNA pair of interest, was amplified using one step RT-PCR with a suitable primer from synthetic RNA controls of indicated SARS-CoV-2 variant. The amplified product was used as target or input for the trans-cleavage assay. A guide pair was defined as selective, if a trans-cleavage assay done with the “Omicron- specific guide” showed 5 fold higher signal in presence of the target amplified from Omicron synthetic RNA control than in the presence of the target amplified from reference synthetic RNA control after 1 hour of the assay, and the trans-cleavage assay done using the “Reference-specific guide” showed 5 fold higher signal in presence of the target amplified from reference synthetic RNA control than in the presence of the target amplified from Omicron synthetic RNA control after 1 hour of the assay. The selectivity of guide pair can be lost at higher concentration of target in the trans-cleavage assays. Hence, in order to make an assay that could distinguish Omicron variant from non-Omicron variant in a sample that has unknown concentration of the RNA, a guide pair is needed that retains selectivity at high input RNA concentration. Accordingly, a test was devised to evaluate if the selectivity of the guide RNA will get compromised at higher input RNA concentrations or not. The input to trans-cleavage assay is a DNA fragment amplified from the input RNA sample using RT-PCR. The maximum amount of the target DNA that can be expected at the end of RT-PCR amplification is equal to primer concentration. About 250 nM primer was used for amplification; hence the highest possible concentration of the amplified product was about 250 nM. About 5 µL of the amplified product was employed in about 50 µL of reaction for trans-cleavage assay, so the highest possible concentration of the target DNA was about 25 nM. So, it was reasonable to believe if a guide pair showed selectivity in the trans-cleavage assay performed in the presence of 30 nM of the synthetic DNA targets, the selectivity would be retained for any amount of input viral RNA present in the unknown sample to be tested. Synthetic DNA templates corresponding to the amplicon region were synthesized as described in materials and methods section. The trans-cleavage assays were done using these synthetic DNA targets (about 30 nM) as input for the selected guide pair to estimate the selectivity at saturating sample concentrations. The guide RNA pair was defined as suitable for the end-point assay if a trans-cleavage assay done with Omicron-specific guide showed more than 5 fold higher signal in the presence of Omicron synthetic DNA target (30nM) than in the presence of the reference synthetic DNA target (30 nM) after one hour of the assay, and trans-cleavage assay done with Reference-specific guide showed more than 5 fold higher signal in presence of Reference synthetic DNA target (30 nM) than in the presence of the Omicron synthetic DNA (30 nM) after one hour of the assay. Panel (b) of Figure 1 shows results of the trans-cleavage assay for the selected guide RNA pair of S and N gene in the presence of about 30 nM of the target DNA concentration. The S gene guide pair that was found suitable recognizes and binds a stretch of sequence that harbors a set of three mutations; Q493R, G496S, and Q498R. The “Omicron-specific” guide RNA of the S gene guide pair is represented by SEQ ID No.1, whereas the “Reference-specific” sgRNA of the S gene guide pair is represented by SEQ ID No.3. The BA.2 variant of Omicron does not harbor the G496S mutation. Interestingly, it was observed that both the Reference-specific and Omicron-specific guide of this guide pair could induce trans- cleavage in the presence of the synthetic DNA template corresponding to BA.2. This made it possible to predict if the Omicron variant was BA.2 or not. The presence of polymorphism in SARS-CoV-2 genes lead to recommendations that for SARS- CoV-2 diagnostic tests should at least include two genes in order to avoid false negative results (Vanaerschot et al., 2020). Hence, ORF1ab was included as the second gene for OmiCrisp and

10) were designed that would give a positive signal for both Omicron and non-Omicron variants of the SARS-CoV-2 standard design rules. ORF1ab allowed confirmation of presence of SARS-CoV-2, irrespective of the specific variant. Furthermore, an initial observation, during clinical validation, was that S gene guide pair gave a lower or undetectable signals at low sample loads as shown in panel b of figure 2, hence compromising the sensitivity of assay of SARS-CoV-2 prediction. To overcome this limitation, it was decided to include N gene as third gene in the assay. A suitable mutation that could be used to discriminate Omicron from non-Omicron variants according to the criteria described previously was identified. Accordingly, a selective guide pair that could be used in end-point assay was designed. This guide pair for N gene targets the region that harbors mutation NΔ31-33; was included in the assay as depicted in panel (c) of figure 1. The “Omicron-specific” sgRNA of the N gene guide pair is represented by SEQ ID No.2, whereas the “Reference-specific” sgRNA of the N gene guide pair is represented by SEQ ID No.4. The above-referred sequences are depicted in the below table (Table 1). Table 1: SEQ ID No. Name Sequence (5'-3') SEQ ID No. 1 SOm UAAUUUCUACUAAGUGUAGAUCGAUCAUAUAGUUUCCGACC SEQ ID No. 2 NOm UAAUUUCUACUAAGUGUAGAUAGAAUGGUGGGGCGCGAUC SEQ ID No. 3 SRef UAAUUUCUACUAAGUGUAGAUCAAUCAUAUGGUUUCCAACC SEQ ID No. NRef UAAUUUCUACUAAGUGUAGAUGAAUGGAGAACGCAGUGG 4 SEQ ID No. 9 ORF1abRef UAAUUUCUACUAAGUGUAGAUAAAAUUACAGAAGAGGUUGG SEQ ID No 10. ORF1abOm UAAUUUCUACUAAGUGUAGAUUAUUAUUUGCUGGUUUAAGU EXAMPLE 2: Primer design Primers that could amplify the regions of interest in S gene, ORF1ab, and N gene in the majority of SARS-CoV-2 isolates were designed. Consensus primers for a group of 23 variants of SARS- CoV-2 that represent major mutation constellations of SARS-CoV-2, were designed using NCBI primer BLAST. In order to estimate the applicability of these primers for Indian isolates of SARS- CoV-2, these primers were aligned with all the complete genome sequences of SARS-CoV-2 Indian isolates deposited at NCBI. The frequency of isolates that have mismatches with primer was analyzed. It was observed that 99% of the isolates had no mis-matches. The sequence of primers and the frequency of mismatches are provided in Table 2. Table 2: Frequency of complete genome sequences of SEQ ID No. Name Sequence (5'-3') Indian isolates with 1 or more mismatches with indicated primers SEQ ID No. 11 Fwd_Sgene GGAAGTCTAATCTCAAACCTTTTGAGAGAG 9/1375 SEQ ID No. Rvs_Sgene AGACTCAGTAAGAACACCTGTGCC 3/1375 12 SEQ ID No. 13 Fwd_Ngene CCTCAGATTCAACTGGCAGTTTCCAG 4/1375 SEQ ID No. 14 Rvs_Ngene AGAGCGGTGAACCAAGACGCA 2/1375 SEQ ID No. 15 Fwd_ORF1ab GAGGACGCGCAGGGAATGGA 14/1375 SEQ ID No. 16 Rvs_ORF1ab TAGTAAGACTAGAATTGTCTACATAAGCAGC 11/1375 SEQ ID No. 17 Fwd_RNaseP CCAGAGACCGACACACGGGA Not Applicable SEQ ID No. 18 Rvs_RNaseP ATGCGAAGAGCCATATCACGGAGGGGATAA Not Applicable EXAMPLE 3: Analytical validation of the assay The complete assay includes two steps. First a fragment of interest of ORF1ab, N, S, and human RNaseP gene was amplified from the sample to be tested using about 5 µL of the sample to be tested in about 70 µL of the reaction volume using multiplexed one step RT PCR as described in materials and methods section. In the second step, the presence of the of amplified fragment of interest of ORF1ab, N, S, and human RNaseP was tested using trans-cleavage assay. The amplified product was used as an input for end-point trans-cleavage assays in the presence of 6 different detection reagents containing the following guide RNAs: 1. ORF1ab (SEQ ID No.9 and SEQ ID 10), 2. SOm (SEQ ID No.1), 3. SRef (SEQ ID No.3), 4. NOm (SEQ ID No.2), 5. NRef (SEQ ID No.4), and 6.RNaseP (SEQ ID 19 UAAUUUCUACUAAGUGUAGAUAAUUACUUGGGUGUGACCCU). Panel (a) of Figure 1 depicts the design of the assay. Step-1 or the multiplexed amplification was carried out in PCR tubes. RT-NTC control is included at this step, in this control nuclease free water is added to the indicated tube instead of a sample. Step-2 or detection step was carried out in a 96-well plate. The columns of the detection plates as depicted in the figure are labeled to show the guide RNA present in corresponding wells and the rows are labeled with the target sample added to the wells of that row. To the rows labelled S1-S6, RT-PCR amplified products of the clinical samples to be tested were added as input target. To the row labeled Det_pos, synthetic target for testing the quality of the detection reagents were added. To the row labeled Det_neg, nuclease free water was added. For each trans-cleavage reaction about 5 µL of the amplified sample was used and the reaction in each detection reagent was done in duplicate, resulting in a total of 12 independent trans-cleavage reactions for each sample to be tested. For analytical validation a known number of copies of a given synthetic RNA control was used as sample to be assayed. As can be seen in panel c of figure 1 when 100 copies of either Omicron or Reference synthetic RNA control samples was used, a detectable signal was observed at the end of the assay; hence the limit of detection of the assay was determined to be at least 100. It was observed Reference synthetic controls samples showed more than 2 times higher signal in the trans-cleavage reactions that had Reference-specific guides (SRef or NRef) than in the trans-cleavage reactions that has Omicron-specific guide of corresponding gene (SOm or NOm), and Omicron synthetic controls samples showed more than 2 times higher signal in the trans-cleavage reactions that had Omicron-specific guides (SOm or NOm) than in the trans-cleavage reactions that had Reference-specific guide of corresponding gene (SRef or NRef). Hence, the N gene and S gene guide pair were able to discriminate the Omicron synthetic RNA from reference synthetic RNA at all the concentration of input RNA tested using the assay. Clinical Validation The first version of the assay referred to as OmiCrisp_v1 did not have N gene guide RNAs. It comprised four sgRNAs; three for SARS-CoV-2: ORF1ab (SEQ ID No. 9), S gene Omicron- specific (SEQ ID No. 1), S gene reference-specific (SEQ ID No. 3), and one for human gene: RNase P gene (SEQ ID No. 19: UAAUUUCUACUAAGUGUAGAUAAUUACUUGGGUGUGACCCU). Hence, the detection step involved 8 independent trans-cleavage reactions that had these guide RNAs in duplicates. OmiCrisp_v1 was used to predict the presence or absence of SARS-CoV-2 and to predict if it was an Omicron variant or non-Omicron variant in RNA samples extracted from nasopharyngeal swabs and the predictions were compared with the sequencing results of these samples. Out of these 50 samples only 46 were detected as SARS-CoV-2 positive by OmiCrisp_v1; therefore, the sensitivity of the kit for detecting SARS-CoV-2 was determined to be about 92 %. Among the 46 samples detected as SARS-CoV-2 positive by OmiCrisp_v1, 40 were identified as Omicron and 6 were identified as non-Omicron. All the samples identified as Omicron by OmiCrisp_v1 were indeed Omicron, the sample identified as “non-Omicron” were Delta (non- Omicron) as per sequencing results. Therefore, the specificity of OmiCrisp_v1 for distinguishing Omicron from non-Omicron variants was determined to be about 100 % (Figure 2(a), 2(b)). OmiCrisp_v1 had a good selectivity to call out Omicron from non-Omicron variants; however, in order to improve the sensitivity to predict SARS-CoV-2 positivity, the data of 4 samples that were falsely identified as SARS-CoV-2 negative was analyzed. It was observed that two of these samples had unambiguously higher signals over no template controls for ORF1ab gene. But because of the S gene signal being low, the samples were identified as negative (Figure 2a). Next, the Ct values of all the four samples were estimated using a commercially available rtPCR kit that targets S gene, N gene, and RdRP (Figure 2(c)). Interestingly in three out of these four samples S gene was not detectable, and the Ct values of the N gene, and RdRp were higher than 32 in these samples. This was indicative of lower viral load or degraded RNA in these samples. Based on the above data, to enable detection at viral lower loads, it was decided that the number of RT-PCR cycles would be increased from 35 to 40 at the amplification step, and one more gene would be included in the assay to minimize the false negative results. As discussed previously, a new guide pair that targets a region on N gene (SEQ ID Nos.2 and 4) was included in version 2 of the OmiCrisp assay. This version of the assay was referred to as Omcrisp_v2. The Omcrisp_v2 assay was validated on a total of 33 RNA samples extracted from nasopharyngeal swabs. This validation was done in a blinded fashion. The results of the OmiCrisp_v2 validation are shown in panel a of Figure 3. Results of the validation are provided in a tabular summary in panel (b) of figure 3. Out of 33 samples 25 samples were SARS-CoV-2 positive and 8 samples were SARS-negative. OmiCrisp_v2 correctly identified the positive sample as SARS-CoV-2 positives and negative samples as SARS-CoV-2 negative. Hence, the specificity and sensitivity of OmiCrisp_v2 in detecting SARS-CoV-2 in this validation study were both 100 %. Out of 25 positive samples, 9 samples were Delta variants of the SARS-CoV-2 virus and OmiCrisp_v2 correctly identified them as non-Omicron. Among the remaining 16 samples that were Omicron, 15 were accurately identified as Omicron. One of the Omicron samples was labeled as ambiguous by OmiCrisp_v2 because it was identified as Omicron with S-gene guide set and as non-Omicron as N-gene guide set. It was therefore concluded that OmiCrisp is well suited for tracking the SARS-CoV-2 Omicron variant in clinical samples. EXAMPLE 4: Impact of choice of target loci As described previously, multiple guide pairs that target different target loci on SARS-CoV-2 genome were designed and after evaluating their experimental performance, the choice was narrowed down to two such pairs that target N gene (SEQ ID Nos.1 and 3) and the S gene (SEQ ID Nos.2 and 4). Some examples of the guide pair that were not considered selective or not suitable for the assay are depicted in Figure 4. Figure 4(i) shows selectivity of guide pair designed to target a region of S gene that harbors G339D mutation. The region of interest was amplified using suitable primer and 5 * 10^5 copies of either Omicron synthetic RNA or Reference synthetic RNA control. The amplified samples were subjected to the trans-cleavage in presence of either Reference-specific guide or Omicron-specific guide. This guide pair was labeled as not-selective because the trans-cleavage reaction done with Reference-specific guide did not show more than 5 times higher signal in the presence of Reference target than in the presence Omicron target, and the trans-cleavage reaction with Omicron-specific guide did not show 5 times higher signal in the presence of Omicron target than in the presence Reference target after about 1 hour of trans-cleavage. Figure 4(ii) shows selectivity of guide pair designed to target a region of S gene that harbors N679K and P681H mutations. The region of interest was amplified using suitable primer and 5 * 10^5 copies of either Omicron synthetic RNA or Reference synthetic RNA control. The amplified samples were subjected to the trans-cleavage in presence of either Reference-specific guide or Omicron-specific guide. Panel a of figure 4 (ii) shows the results. This guide pair was labeled as selective because trans-cleavage done with Reference-specific guide showed more than 5 times higher signal in the presence of Reference target than in the presence Omicron target, and the trans- cleavage done with Omicron-specific guide showed more than 5 times higher signal in the presence of Omicron target than in the presence Reference target after about 1 hour of trans- cleavage. Further, the suitability of the guide pair for the assay was tested by using about 30 nM of purified DNA target input target to the trans-cleavage assay. The results are shown panel c of the figure 4 (ii). This guide pair was not considered suitable for the assay because the tans-cleavage done at higher target concentration showed that the guide pair could not distinguish between Omicron and non-Omicron variants at higher sample concentrations. Figure 4(iii) shows selectivity of the S493 guide pair that targets the region of S gene harboring, Q493R, 496S, and 498R mutations, and N gene guide pair that targets the region of S gene that harbors NΔ31-33 mutation. It can be seen that for both of these guide pairs, Reference-specific guide showed more than 5 times higher signal in the presence of Reference target than in the presence Omicron target and Omicron-specific guide showed 5 times higher signal in the presence of Omicron target than in the presence of Reference target after about 1 hour of trans-cleavage done in presence of about 30 nM target DNA. From the results, it was inferred that the S493 and N gene guide pairs can be used to discriminate Omicron strains from non-Omicron strains in an end-point assay with unknown viral concentration. EXAMPLE 5: Impact of buffer composition The trans-cleavage assay performed with a control guide RNA in presence of various concentrations of control DNA target: low (0.03 nM), medium (0.3 nM), and high (3 nM). The assay was performed in presence of the following buffer compositions: Buffer 1: Tris-HCl (10 mM), MgCl2 (10 mM), NaCl (50 mM), BSA (100 µg/mL) pH 7.5 at 25 °C. Buffer 2: Tris-HCl (10 mM), MgCl2 (10 mM), NaCl (50 mM) pH 7.5 at 25 °C. Buffer 3: Tris-HCl (10 mM), MgCl2 (10 mM), NaCl (50 mM), recombinant albumin (100 µg/mL) pH 7.9 at 25 °C. Buffer 4: Tris-acetate (20 mM), Potassium Acetate (50 mM), Magnesium Acetate (10 mM), BSA 100 µg/mL pH 7.9 at 25 °C. Buffer 5: Tris-acetate (20 mM), Potassium Acetate (50 mM), Magnesium Acetate (10 mM), recombinant albumin (100 µg/mL) pH 7.9 at 25°C. Results depicting the impact of the choice of buffer composition are provided in Figure 5. It was found that the trans-cleavage activity is dependent on the buffer composition. Although at higher DNA concentrations the trans-cleavage was detected in all the buffers, the impact of buffer in efficiency of trans-cleavage became apparent at medium and low target concentration. Buffers 1 and 4 were found to be optimal for the trans-cleavage-based assay of the present disclosure. EXAMPLE 6: Detection in sewage sample Testing limit of detection in presence of sewage matrix

of template RNA was tested in the presence (plus sewage) and absence (minus sewage) of sewage in the background. Nucleic acid extracted from ten SARS-CoV-2 negative samples were pooled for matrix testing in “plus sewage”. For “minus sewage” samples the pooled nucleic acid from sewage samples was replaced by nuclease free water.100 and 1000 copies of Non-Omicron (Ref) and Omicron (BA.1) synthetic templates were spiked into the plus and minus sewage matrix. RT-NTC control samples had no synthetic templates. The trans-cleavage assay was performed as per the protocol of the present disclosure. Results of the above experiment are depicted in Figure 6.100 and 1000 copies of both the Non- Omicron and Omicron synthetic templates were detected in the absence and presence of background RNA from sewage samples. The signal for the N gene target was higher compared to the S gene target for both types of variant templates. Interestingly, the signal from all 3 targets (ORF1ab, S gene and N gene), though clearly above the background threshold, were dampened in the presence of nucleic acid from sewage samples. A signal from the human gene control (RNase_P) was detected in all samples in the presence of nucleic acids from sewage, but not in the absence. EXAMPLE 7: Detection in sewage sample Testing Limit of detection in a mixed sample the limit of detection in where more than one variant maybe present in the same sample, simulated mixed samples were created by pooling equal copies of 3 different variants synthetic templates: Wuhan (Non-Omicron), BA.1 (Omicron) and BA.2 (Omicron). Total copies tested were 300, 3000 and 30,000 and these were spiked into the pooled nucleic acid extracted from SARS-CoV-2 sewage samples. The trans-cleavage assay was performed as per the protocol of the present disclosure. Results of the above experiment are depicted in Figure 7. Both Non-Omicron and Omicron variants were detectable at levels as low as 300 copies of the target, clearly above the background signal. The signal was found to increase at 3000 and 30000 copies. As expected, signal from both S gene and N gene were detectable. Individual synthetic templates for non-Omicron and Omicron were detected at 100 and 200 copies respectively when the total number of target copies was 300. EXAMPLE 8: Detection in sewage sample Testing Limit of detection in a mixed sample where the variant of interest is at a low fraction (1:5, 1:100)

further test the limit of detection when the representation of one variant is significantly lower than the other variants, the above experiment was modified. The Omicron variant template BA.1 was spiked at 500 copies while the non-Omicron was represented 5, 25 and 100 fold higher at 2500, 12500 and 50000 copies. The templates were spiked into extracted nucleic acid from SARS-CoV-2 negative sewage samples. The trans-cleavage assay was performed as per the protocol of the present disclosure. Results of the above experiment are depicted in Figure 8. The data indicated that 500 copies of the virus variant were clearly detected above background even when present at 1/100th part. EXAMPLE 9: Detection in sewage sample Testing specificity of the SARS-CoV-2 detection guides in samples previously tested negative for SARS-CoV-2

Nucleic acids from 6 sewage samples were selected that were previously negative for SARS-CoV- 2 by RT-PCR assay. The Ct value for the amplification of the human gene control in these samples ranged from 30-35 indicating that these were mid to low quality samples. The trans-cleavage assay was performed as per the protocol of the present disclosure. Results of the above experiment are depicted in Figure 9.3 of the samples (sample 2, 3, 5) showed that the human gene signal was below threshold and hence could be considered in the analysis. In samples 1, 4 and 6, clear signal from the human gene was detected above the threshold. No signal from any of the SAR-CoV2 target genes were detected above the background threshold. When the experiment was repeated on a new set of negative samples, one sample (Sample 3) did not give signal for the human gene. The human gene signal was detected in all the remaining samples. No signal from any of the SAR-CoV-2 target genes were detected above the background threshold. Specificity of guides of SARS-CoV-2 in pooled nucleic acids from 10 negative samples.

6(a), 7 and 8. In

pooled nucleic acid from 10 RT PCR negative sewage samples were used as RT-PCR negative control for SARS-CoV-2. The pooled sample was subjected to testing with the assay for SARS- CoV-2 targets. Except for the human gene signal, no SARS-CoV-2 target specific signal was detected in these RT-NTC samples. Specificity of CRISPR assay for the detection of SARS-CoV-2 non-Omicron vs Omicron variants. The specificity of distinction of Omicron variants from the non-Omicron variants was detected in the experiment depicted in Figure 1(b). The targets on S gene and N gene were amplified at saturating / highest concentrations of synthetic templates possible in the RT-PCR assays. Post amplification, the CRISPR assays with variant specific guides could distinguish the Omicron variants (using NOm and SOm guides) from the non-Omicron variants (using NRef and SRef guides). EXAMPLE 10: Field validation of SARS-CoV-2 detection and distinguishing Omicron variants from the non-Omicron variants Testing on good quality retrospective samples:

field samples that were confirmed to be Omicron positive by RT-PCR. Samples were chosen such that the Ct value of the N gene in the RT-PCR assay were found to be 25-28 at the time of testing. These samples were selected from January 2021. Results of the above experiment are depicted in Panel (a) of Figure 10. All samples were SARS- CoV-2 positive as per the CRISPR assay. The variant was identified as Omicron (which overlapped with the time frame in which the variant appeared). Human gene control was detected in all the samples by the CRISPR assay. Testing on good, medium and poor-quality retrospective positive sample:

found to be SARS- CoV-2 positive by using RT-PCR assay. These samples were selected from the time frame of December 2021, January and February 2022. The samples were classified as good, medium or poor quality, based upon the Ct value observed for the N gene (range 26.1-35.6), in the RT-PCR assay. Results of the above experiment are depicted in Panel (b) of Figure 10. It was seen that the OmiCrisp assay identified all samples as SARS-CoV-2 positive. Though human gene could not be detected in 2 samples (10 and 12), the signal from the SARS-CoV-2 target signals were strong enough for calling the samples positive by the CRISPR assay and also variant identification. All samples from January and February (Ct value for N gene were 26.1 - 34.64) were unambiguously identified as Omicron variant. The single sample from December 2021 (Sample 18) was identified as non-Omicron. Testing on retrospective positive samples of medium quality in a time frame where non-Omicron

A series of experiments was conducted on medium quality positive samples (Ct value of N gene between 30 and 32). The samples were selected from December 2021 when the non-Omicron variant (Delta) was in circulation.18 independent samples were tested. Results of the above experiment are depicted in Panel (c) of Figure 10. The data depicts unambiguous detection of SARS-CoV-2 and identification of the non-Omicron variants in the sample. The foregoing description fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the general concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein, without departing from the principles of the disclosure. Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

Claims

We claim: 1) CRISPR sgRNA(s) selected from a group of sequences represented by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4. 2) The CRISPR sgRNA(s) as claimed in claim 1, wherein SEQ ID Nos. 1 and 3 bind to a target in the gene encoding S protein of SARS-CoV-2; and wherein SEQ ID Nos.2 and 4 bind to a target in the gene encoding N protein of SARS-CoV-2. 3) The CRISPR sgRNA(s) as claimed in claim 1, wherein SEQ ID Nos. 1 and 3 bind to a target in the gene encoding S protein of SARS-CoV-2 represented by SEQ ID Nos.5 and 7, respectively and wherein SEQ ID Nos.2 and 4 bind to a target in the gene encoding S protein of SARS-CoV-2 represented by SEQ ID Nos.6 and 8, respectively. 4) The CRISPR sgRNA(s) as claimed in claim 3, wherein the S protein target of SEQ ID No. 1 comprises one or more mutations selected from Q493R, G496S, and Q498R; wherein the N protein target of SEQ ID No. 2 comprises mutation NΔ31-33; wherein the S protein target of SEQ ID No.3 is a non-Omicron sequence from SARS-CoV-2; and wherein the N protein target of SEQ ID No.4 is a non-Omicron sequence from SARS-CoV-2. 5) The CRISPR sgRNA(s) as claimed in claim 1, for use in detecting SARS-CoV-2 in a sample. 6) The CRISPR sgRNA(s) as claimed in claim 1, for use in detecting an Omicron variant of SARS-CoV-2 in a sample; and/or subvariant of the Omicron variant selected from subvariants BA.1, BA.2 and BA.3. 7) A method of detecting SARS-CoV-2 in a sample, comprising contacting the sample with one or more of the CRISPR sgRNA(s) as claimed in claim 1 in presence of Cas12a nuclease or orthologs thereof, a reporter system for trans-cleavage activity of Cas12a or orthologs thereof and optionally, buffer and/or water. 8) The method as claimed in claim 7, wherein the reporter system is in the format of F-(N)n- Q or Q-(N)ⁿ-F; wherein F is a fluorescent reporter molecule selected from a group comprising SYT09, fluorescein, rhodamine, rhodoamine600, R-phycoerythrin, and Texas Red; wherein N is selected from A, G, T, C, rA, rG, rT and rC; wherein Q is a quencher such as but not limited to black hole quencher (BHQ), Iowa black and 4- (dimethylaminoazo)benzene-4-carboxylic acid (DABCYL) and wherein n ranges from about 6 to about 15. 9) The method as claimed in claim 7, wherein the method is implemented as an in-vitro assay; wherein the in-vitro assay is performed in a multi-well plate, multi-strip tube or individual tube(s); wherein each well comprises a different CRISPR sgRNA along with Cas12a nuclease, reporter system and optionally, buffer and/or water. 10) The method as claimed in 9, wherein the in-vitro assay further employs additional sgRNA(s) against a target in the SARS-CoV-2 genome different from that targeted by any of the SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4. 11) The method as claimed in 10, wherein the additional sgRNA targets ORF1ab gene in SARS-CoV-2; wherein the additional sgRNA(s) has sequence(s) represented by SEQ ID Nos.9 and/or 10. 12) The method as claimed in claim 7, wherein the method detects Omicron variant of SARS- CoV-2 in a sample; and/or wherein the method detects subvariants of the Omicron variant selected from subvariants BA.1, BA.2 and BA.3. 13) The method as claimed in claim 7, wherein the sample is a biological sample or an environmental sample. 14) The method as claimed in claim 7, wherein the sample is subjected to amplification prior to contacting with the sgRNA(s); wherein the amplification is performed by RT-PCR; and/or wherein the application is performed using primer(s) selected from a group comprising SEQ ID Nos.11-18. 15) The method as claimed in claim 7, wherein the method has analytical limit of detection ranging from about 100 copies to about 500 copies. 16) The method as claimed in claim 7, wherein the method has specificity ranging from about 80% to about 100%; and/or sensitivity ranging from about 80% to about 100%. 17) A kit comprising the CRISPR sgRNA(s) as claimed in claim 1; Cas12a nuclease or orthologs thereof; reporter system for trans cleavage activity of Cas12a nuclease or orthologs thereof; and optionally, water, buffer, additional sgRNA(s) against a target in the SARS-CoV-2 genome different from that targeted by any of the SEQ ID No. 1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, the primers as defined in claim 14, reagents for amplification, a multi-well plate or multi-strip tube or individual tube(s), and an instruction manual. 18) The kit as claimed in claim 17, wherein the sgRNAs are contained in the multi-well plate or multi-well plate multi-strip tube or individual tube(s); and wherein the multi-well plate or multi-well plate multi-strip tube or individual tube(s) comprising the sgRNAs constitutes an assay device.