A buffer for a CAS protein and a method of using thereof

Abstract

Disclosed is a buffer for enhancing enzymatic cleavage activity of a Cas protein, 5 comprising one or more components selected from the group consisting of: a salt selected from the group consisting of monopotassium phosphate (KH₂PO₄), sodium formate (HCOONa), calcium acetate (Ca(C₂H₃O₂)₂), sodium acetate (CH₃COONa), ammonium sulfate ((NH₄)₂SO₄), potassium thiocyanate (KSCN), calcium chloride (CaCl2), sodium chloride (NaCl), Nickel(II) nitrate (Ni(NO3)2), lithium sulfate (LiSO4); an organic solvent 0 selected from the group consisting of Tween 20, Dithiothreitol (DTT), Phenylmethylsulfonyl fluoride (PMSF), 2-Methyl-2,4-pentanediol (MPD), acetone, dimethylsulfoxide (DMSO), ethylene glycol, methanol, 2-propanol, EDTA, urea; and a protein which is casein. Also disclosed is a detection kit for a nucleic acid, comprising the buffer as disclosed herein, a Cas protein and a crRNA. Further disclosed is a method 5 of detecting a nucleic acid in a sample by CRISPR-cas, using the detection kit as disclosed herein.

Description

A BUFFER FOR A CAS PROTEIN AND A METHOD OF USING THEREOFFIELD OF THE INVENTION

[0001] The present invention belongs to the field of buffer for Cas protein to enhance its enzymatic cleavage activity. The present invention also relates to the field of nucleic acid detection by CRISPR-Cas using said buffer.BACKGROUND

[0002] Recently novel Cas 13 enzymes which can be used in clustered regularly interspaced short palindromic repeats (CRISPR / Cas)-based methods for detecting nucleic acids were disclosed in PCT / SG2022 / 050859. This approach has gained prominence as viable alternatives to traditional methodologies. These approaches are characterized by their independence from specialized instrumentation, alongside their simplicity, sensitivity, specificity, affordability, and rapidity. Additionally, they possess the capacity for multiplex pathogen detection within a single assay. The novel aspect of these methodologies lies in leveraging the collateral cleavage activity of Cas proteins, facilitating the transformation of target nucleic acid sequences into observable signals. When a Cas protein such as Casl3, in complex with CRISPR RNA (crRNA), identifies its target RNA, it becomes activated. This activation triggers Cas 13 to indiscriminately cut the quenched fluorescent ssRNA reporter molecules. As a result of this cleavage, the fluorescent dye is released, producing a detectable signal.

[0003] There is a need to understand how the efficiency of enzymatic cleavage by Cas 13 can be influenced by a variety of reaction buffer conditions, in order to find an optimal buffer composition, and optimize the trans-cleavage activities of Cas 13. To refine the Cas 13 reaction buffer, various commercially available buffers were initially evaluated to define the best buffer for Cas 13 trans-cleavage activity. Several published or commercially available buffer systems that are marketed or commonly used for Casl3- based diagnostics, including buffers from IDT (used in SHERLOCK assays) and other vendors offering CRISPR diagnostic kits, were evaluated. These buffers were primarily optimized for earlier and well-characterized Casl3 enzymes such as LwaCasl3a or LbuCasl3a. However, these commercial buffers often did not support optimal activity for the inventors' novel Cas 13 enzymes. Specifically, they resulted in reduced collateralcleavage efficiency, delayed reaction kinetics, and lower overall signal intensity. This highlighted the need for a customized buffer formulation, as commercial solutions may not be universally compatible with newly developed or less-characterized Casl3 variants. Enzymatic cleavage activities are widely recognized to be influenced by numerous reaction conditions such as ion types, salt concentrations, and other factors. Consequently, a thorough investigation of the variables impacting the trans-cleavage activities of Cas 13 was conducted. By systematically analysing and optimizing these reaction conditions, the optimal buffer that maximizes Cas protein activity, such as Cas 13 activity was able to be identified and established.SUMMARY

[0004] In one aspect, the present disclosure refers to a buffer for enhancing enzymatic cleavage activity of a Cas protein, comprising one or more components selected from the group consisting of: a salt selected from the group consisting of monopotassium phosphate (KH2PO4), sodium formate (HCOONa), calcium acetate (Ca(C2H3O2)2). sodium acetate (CH3COONa), ammonium sulfate ((NH4)2SO4), potassium thiocyanate (KSCN), calcium chloride (CaCl2). sodium chloride (NaCl), Nickel(II) nitrate (Ni(NO3)2), lithium sulfate (LiSO4); an organic solvent selected from the group consisting of Tween 20, Dithiothrcitol (DTT), Phenylmethylsulfonyl fluoride (PMSF), 2-Methyl-2,4-pentanediol (MPD), acetone, dimethylsulfoxide (DMSO), ethylene glycol, methanol, 2-propanol, EDTA, urea; and a protein which is casein.

[0005] In another aspect, the present disclosure refers to a detection kit for a nucleic acid, comprising:(a) the buffer as disclosed herein;(b) a Cas protein selected from the group consisting of Casl3d, Casl3a, Casl3b, Casl3c, Casl2a, Casl2b, Casl2c, Casl2d, and Casl2f; and(c) a crRNA configured to target the nucleic acid and form a complex with the Cas protein.

[0006] In another aspect, the present disclosure refers to a method of detecting a nucleic acid in a sample by clustered regularly interspaced short palindromic repeats andCRISPR associated protein (CRISPR-cas), using the detection kit as disclosed herein, the method comprising: incubating the sample containing the nucleic acid with the Cas protein and the crRNA, wherein when the nucleic acid is present in the sample, enzymatic cleavage activity of the Cas protein is induced to generate a detectable signal when the probe nucleic acid is cleaved; wherein the buffer enhances the enzymatic cleavage activity of the Cas protein.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0008] Figure 1 shows ionic influence on Casl3-Cdl4 activity under varying salt conditions, a. the influence of KH2PO4, CdSO4, Co(NO3)2, HCOONa, Ca(C2H3O2)2, CH3COONa, b. the influence of (NH4)2SO4), AlCl3, KSCN, FeSO4, CuSO4, LiSO4, c. the influence of Ah(SO4)3, ZnCl2, CaCl2, NaCl, K2SO4. The fluorescence signal measured after 5 min has been presented here. Values arc shown in the graphs as means ± SD (n = 3). The dotted line in the graph represents the baseline activity, as determined by the control reaction buffer.

[0009] Figure 2 shows the influence of organic compounds on Casl 3-Cd14 activity, a. the influence of DTT, urea, EDTA and PMSF. b. the influence of ethylene glycol, methanol, DMSO, Tween 20, 2-Propanol, acetone, and MPD. The fluorescence signal measured after 5 min has been presented here. Data are represented as mean values with standard deviation (mean ± SD, n = 3).

[0010] Figure 3 shows the effect of casein on the collateral cleavage activity of Casl3- Cdl4. The fluorescence signal measured after 5 min has been presented here. Values are shown in the graphs as means ± SD (n = 3).

[0011] Figure 4 shows ionic influence on Casl3-Cdl4 activity in SARS-CoV-2 detection, a. the influence of LiSO4and Ah(SO4)3. b. the influence of (Ni(NO3)2a)nd ZnCl2c. the influence of CaCl2and NaCl. The fluorescence signal measured after 5 min has been presented here. Values arc shown in the graphs as means ± SD (n = 3).

[0012] Figure 5 shows the influence of organic compounds on Casl3-Cdl4 activity, a. the influence of urea and PMSF. b. the influence of EDTA and DMSO. c. the influence of Tween 20 and acetone, d. the influence of MPD and methanol, e. the influence of propanol and ethylene glycol. The fluorescence signal measured after 5 min has been presented here. Data arc represented as mean values with standard deviation (mean ± SD, n = 3).

[0013] Figure 6 shows the impact of Casein on Casl3-Cdl4 activity in SARS-CoV-2 detection. The fluorescence signal measured after 5 min has been presented here. Values are shown in the graphs as means ± SD (n = 3).

[0014] Figure 7 shows real-time fluorescence signals of Casl3-Cdl4 at varying SARS-CoV-2 DNA concentrations. Real-time PCR signals were monitored different target DNA concentrations (0.0015, 0.0075, 0.015, 015, and 1.5 copies / μL). The fluorescence was measured in 1 min intervals for a total of 60 min. The measurement of real-time background subtracted fluorescence output are shown in the graphs as means± SD (n = 3).

[0015] Figure 8 shows enhancement of Casl3-Cdl3 and Casl3-Cdl5 activity by Tween 20, MPD, and Cascin. Fluorescence intensity was measured to assess the collateral cleavage activity of Casl3-Cdl3 and Casl3-Cdl5 in the presence of Tween 20, MPD, and Casein. The fold increase in fluorescence signal relative to the no-additive control is shown. Tween 20 and Casein exhibited the highest enhancement effect on Casl3-Cd15, increasing activity by 10.4-fold and 11.2-fold, respectively. Data represent the mean ± standard deviation (n = 3).DETAILED DESCRIPTION

[0016] To enhance enzymatic efficiency of a Cas enzyme such as Casl3 and Casl2 for collateral cleavage and detection of nucleic acids, such as nucleic acids of pathogens, novel compounds (salts, organic solvents and proteins) were introduced into the enzyme reaction buffer, presenting a remarkable increase in enzyme collateral cleavage activity.

[0017] In one aspect, the present disclosure refers to a buffer for enhancing enzymatic cleavage activity of a Cas protein, comprising one or more components selected from the group consisting of:a salt selected from the group consisting of monopotassium phosphate (KH2PO4), sodium formate (HCOONa), calcium acetate (Ca(C2H3O)2), sodium acetate (CH3COONa), ammonium sulfate ((NH4)2SO4), potassium thiocyanate (KSCN), calcium chloride (CaCl2), sodium chloride (NaCl), Nickel(II) nitrate (Ni(NO3)2), lithium sulfate (LiSO4); an organic solvent selected from the group consisting of Tween 20, Dithiothrcitol (DTT), Phenylmethylsulfonyl fluoride (PMSF), 2-Methyl-2,4-pentanediol (MPD), acetone, dimethylsulfoxide (DMSO), ethylene glycol, methanol, 2-propanol, EDTA, urea; and a protein which is casein.

[0018] The technical aspects of the present invention that distinguish it from existing technologies include the precise selection and addition of above-mentioned components for the first time to the Cas enzyme (such as Casl3) reaction buffer. Each of these compounds has been individually proven to enhance collateral cleavage activity of the Cas enzyme (such as Cas 13), and therefore, its capability for target nucleic acid detection, for example pathogen detection. The unique features and benefits of the present invention in comparison to existing technologies are:• Enhanced Enzyme Activity : The individual addition of each compound has resulted in a substantial increase in the Cas enzyme (such as Casl3 and Casl2) collateral cleavage activity, demonstrating the present buffer composition's effectiveness.• Customized Buffer Composition: The deliberate choice and ratio of salts, solvents, and protein in the buffer are tailored to not only augment the Cas enzymatic activity but also to ensure the stability and efficiency of the Cas enzyme across a broader range of conditions.• Versatility in Application: The enhanced activity of the Cas enzyme (such as Cas 13 and Cas 12) by the present compounds expands the potential applications, particularly in the field of pathogen detection, where rapid and efficient detection of pathogen can be critical.• Increased Sensitivity for Pathogen Detection : The higher activity levels obtained could lead to increased sensitivity in diagnostic tests that rely on the Cas enzyme (such as Casl3 and Casl2) collateral cleavage activity, enabling the detection of pathogens at lower concentrations than currently possible.• Cost-Effectiveness: By increasing the specific activity of the Cas enzyme (such as Casl3 and Casl2), less enzyme may be required for the same or enhanced effect, leading to reduced costs for industrial-scale applications.• Adaptability: The improved activity under different conditions suggests that the Cas enzyme (such as Casl3 and Cas 12) could be adapted for use in a variety of environments, which is particularly beneficial for field-based pathogen detection where laboratory conditions cannot be replicated.• Synergistic effects of the buffer components : The synergistic combination of these compounds, which have individually shown to amplify activity, yields a cumulative increase potentially exceeding current level of activity, enhancing enzymatic efficiency significantly.• Broader applicability to a variety of Cas enzymes: The disclosed compounds which enhance the activity of Casl3-Cdl3, Cdl4 and Cdl5 enzymes. While the present experimental data demonstrate enhancement of Casl3 enzymatic activity through the addition of novel buffer components, it can be reasonably anticipated that similar improvements may extend to Cas 12 enzymes. This expectation is based on several shared mechanistic and structural features between Cas 13 and Cas 12 systems. Both enzymes are RNA-guided nucleases that require precise buffer conditions to maintain optimal tertiary conformation and catalytic activity. Furthermore, the identified compounds act through mechanisms such as stabilizing protein -RNA interactions, enhancing target binding, or facilitating ion- dependent conformational changes — features that arc also critical for Cas 12 function. Given these mechanistic similarities, it is plausible that these novel buffer additives will likewise enhance Cas 12 activity. This potential crossapplicability supports the broader utility of the present buffer system across the CRISPR-Cas enzyme family.

[0019] In general, the CRISPR system (Clustered Regularly Interspaced Short Palindromic Repeats) is a powerful tool for gene editing that allows scientists to precisely alter DNA sequences and modify gene function. It was originally discovered as a natural defense mechanism in bacteria and archaea, where it helps these organisms fend off viral infections. CRISPR works via the following steps: (1) Recognition: When a virus invades a bacterial cell, the bacterium incorporates a segment of the viral genetic material —typically DNA, but in some cases RNA (converted to DNA by reverse transcription) — into its own genome at the CRISPR locus. This segment is known as a "spacer." (2) Transcription: The CRISPR locus is transcribed into a long RNA molecule, which is then processed into shorter "guide RNAs" (gRNAs) that include sequences complementary to the viral DNA or RNA. (3) Targeting: The guide RNA forms a complex with a CRISPR- associated (Cas) protein. This complex can recognize and bind to the corresponding viral DNA and RNA sequence. (4) Cleavage: The Cas protein acts as a molecular scissor, cutting the viral DNA and RNA at the targeted location, thereby neutralizing the threat. Applications of CRISPR system includes: (1) Gene Editing: CRISPR can be used to add, remove, or alter specific DNA and RNA sequences in the genome of living organisms. This has vast implications for research, medicine, and agriculture. (2) Gene Therapy: CRISPR is being explored as a potential treatment for genetic disorders by correcting mutations at the DNA level. (3) Functional Genomics: Researchers use CRISPR to study gene function by creating targeted mutations and observing the resulting phenotypes. In the context of the present disclosure, CRISPR is used to detect target nucleic acid of a particular pathogen for pathogen or disease detection.

[0020] Cas 13 is an RNA-targcting enzyme that belongs to the CRISPR family, specifically the Type VI CRISPR-Cas systems. It is comprised of subtypes VI-A, VI-B, VI-C, and VI-D and also known as C2c2 or CasRx (type VI-D). Unlike the well-known Cas9, which targets DNA, Casl 3 specializes in targeting and cleaving single-stranded RNA. This makes it a powerful tool for RNA manipulation and has opened up exciting possibilities in biotechnology and medicine. Cas 13 enzymes are classified into several subtypes based on their structural and functional differences. The diverse Cas 13 family contains at least four known subtypes, including Casl3a (formerly C2c2), Casl3b, Casl3c, and Casl3d. All known Casl3 family members contain two HEPN domains which confer RNase activity. Cas 13s function similarly to Cas9, using a ~64-nt guide RNA to encode target specificity. The Cas 13 protein complexes with the guide RNA via recognition of a short hairpin in the crRNA, and target specificity is encoded by a 28 - 30-nt spacer that is complementary to the target region.

[0021] The CRISPR-Casl3 system has a target-specific nucleic acid cleavage activity, and two conditions arc required to exhibit this target-specific nucleic acid cleavage activity: (1) Recognition of a Specific Nucleotide Sequence: The Casl3 protein mustrecognize a nucleotide sequence of a certain length in the nucleic acid. (2) Complementary Binding of the Guide RNA: The guide RNA (crRNA) must have a sequence that can complementarily bind to the target sequence around the recognized nucleotide sequence. This binding ensures that the Casl3 protein is directed to the correct location on the nucleic acid. When the above two conditions arc satisfied, 1) the Casl3 protein recognizes the nucleotide sequence of a certain length, and 2) the guide RNA complementarily binds to a portion of the sequence surrounding the nucleotide sequence of the certain length, nucleic acid cleavage activity is exhibited.

[0022] In addition to programmable RNase activity, all Casl3s exhibit collateral cleavage activity after recognition and cleavage of a target RNA, leading to non-specific degradation of any nearby single- stranded RNA molecules regardless of complementarity to the spacer. Properties of Casl3 make it well suited for nucleic acid detection. When the enzyme recognizes its target in vitro, it becomes activated and promiscuously cleaves RNA species in solution. Cas 13-based detection is specific, and can be tuned for singlenucleotide distinction at any position on the target. This secondary cleavage activity can be utilized to detect the presence of a target nucleic acid in a sample, making the CRISPR- Casl3 system a powerful tool for diagnostic applications.

[0023] Casl2, also known as Cpfl (type V-A) or C2cl (type V-B), is a CRISPR- associatcd protein that belongs to the Type V CRISPR-Cas systems. It is an RNA-guidcd endonuclease that targets and cleaves double-stranded DNA. Casl 2 is a compact and efficient enzyme that creates staggered cuts in dsDNA. Cas 12 processes its own guide RNAs, leading to increased multiplexing ability. Cas 12 has also been engineered as a platform for epigenome editing, and it was recently discovered that Cas 12a can indiscriminately chop up single- stranded DNA once activated by a target DNA molecule matching its spacer sequence. This property makes Cas 12a a powerful tool for detecting tiny amounts of target DNA in a mixture. The Cas 12 protein requires only the crRNAs to create an efficient cut at ssDNA and dsDNA. Cas 12 protein contains the RuvC and nuclease lobe (NUC) domains for cleavage activity. Like Cas9, Cas 12 encounters a potential target site beside a PAM sequence. Once Cas 12 starts encountering, it initiates R-loop, which forms base-pair hybridization between the crRNA and the target DNA strand. During this step, Cas 12 matches the <17 bp of the target sequence and leads to an R-loop formation. Once R-loop is formed, the Cas 12 protein uses its active RuvC domainto cleave the non-target strand with the help of the PAM sequence. Cas 12 has been widely used in genome editing, diagnostics (like the DETECTR system for detecting pathogens), and other biotechnological applications. Subtypes of Cas 12 enzymes include Cas 12a (Cpfl) known for creating staggered cuts (sticky ends) in DNA, making it useful for precise genome editing. Other subtypes of Casl2 include Casl2b, Casl2c, Casl2d, Casl2e and Casl2f. The CRISPR-Casl2 system has a collateral cleavage activity in addition to the target-specific nucleic acid cleavage activity. When the CRISPR-Casl2 system recognizes and binds to a target nucleic acid, it activates the Cas 12 protein, which then indiscriminately cleaves single-stranded nucleic acids in the vicinity. This secondary cleavage activity can be utilized to detect the presence of a target nucleic acid in a sample, making the CRISPR-Casl2a system a powerful tool for diagnostic applications.

[0024] CRISPR detection is generally divided into 2 parts. The first step is usually the amplification of a target sequence, which specifically comprises the steps of amplifying target nucleic acid by utilizing the technologies of PCR, RT-PCR, RPA, LAMP and the like; the second step is a trans-cleavage reaction of the Cas protein: adding a reporter, such as a non-specific single- stranded DNA or RNA reporter (ssDNA-reporter or ssRNA- reporter) with a fluorescent emitting group at one end and a fluorescent quenching group at one end into the system, and exciting the cleavage activity of the single-stranded DNA or RNA fluorescent reporter after the Cas protcin-crRNA complex is combined with the target DNA, thereby generating a free fluorescent emitting group and emitting detectable fluorescence. In addition to this, CRISPR-Cas detection can be performed in a one-pot system, where target amplification, CRISPR-based recognition, and signal detection occur within a single reaction vessel without the need for multiple handling steps.

[0025] To enhance the sensitivity and efficiency of the target nucleic acid detection, superior trans-cleavage activity of the Cas protein is desired, and this can be achieved or aided by the buffer disclosed herein.

[0026] The present disclosure discloses a buffer for enhancing enzymatic cleavage activity of a Cas protein. In one example, the Cas protein is a Casl3 protein selected from the group consisting of Casl3d, Casl3a, Casl3b, and Casl3c. In another example, the Cas protein is a Casl2 protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d, and Casl2f. The buffer has wide applications to enhance enzymaticcleavage activity of various Cas proteins as disclosed herein which possess collateral cleavage activity.

[0027] As used herein, Cdl3, Casl3-Cdl3, or Casl3d-Cdl3 are interchangeable terms. As used herein, Cdl4, Casl3-Cdl4, or Casl3d-Cdl4 are interchangeable terms. As used herein, Cdl5, Casl3-Cdl5, or Casl3d-Cdl5 arc interchangeable terms. Cdl3 Cdl4, Cdl5 refer to Casl3 protein variants. All of these Casl3 variants can be used in the context of CRISPR technology for precise genome editing, diagnostics, and other biotechnological applications.

[0028] In one example, the Casl3 protein is Casl3-Cdl3, which has an amino acid sequence of SEQ ID NO: 2 as shown in Table 1:

[0029] VSKNDNIKSKAKALGLKSTFQVGDEVVMTSFGKGNKAIVEKIVRGTDVQSVPTEPNFSAEIEGKKFDLVGR AHIQTKSDNPQYSKKRTGDDMIGAKAALEKRFFGGTFDDNIHIQLAYNVLDIEKILSVHINNIVYTLNNIRRKDNAEDDDFIG YMSTRNDYDTFIEPRKHNISEDAAKSIDKSRASFEEYLQPPVKDCLHYFGNTFFAPREIEKTYTDNRGFERKKKVTVNSLI DEKEIYYIFALLGGLRQFCTHDNKSGRNWLYSLEENGINSDAKAVLDKYYNSAVARIDESFVDNASKTNFKLIFNAMDVSD EALQDNIAKAFYKFTVCKSFKNMGFSIKKLREQLLELPEYEGLKDKHYDSVRSKLYQIMDFVIYLSFKDEKFKKDNEEKINN IVNELRATLNEEDKGRVYASYAERMKSELKPAIARLKSDIDKIKDSRVKEFELDASVKYRLSKVVESVRLKDRATYFTKLIY LTTLFLDGKEINDLLTTLIHQFENIASFIDVMNDRGIDCRFSDGYKLFESSKQIAFELRNVNSFARMTRTSKDDENATHMMY IDAAEILGTDYTEEQIEEHLNLEKKRMIPGTKKADMNFRNFIINNVIKSSRFNYLVRYSNPKKIRALADNEGVIRFVLGELPD AQIDRYTLLCGFNPDADRQEKTDKLAKAITGLRFNDFENVKQGANTEGESQESIDKAQKQGLISLYLTVLYLLTKNLVYVN SRYFLAFHCLERDAQLLGSGAGHHEPYVALTQRFINEDKLNEHACEYLKTNIANSDEYTIRIFRNNAAHLSAVRNANLYID KLKEFKSYYEIYHFLSQENIYGKYCVDKKYVTADENGSKTISVKISKDYCPQVYIDKSLEYFDKLNKYGTYCKDFTKALNSP FGYNLARYKNLSIEGLFDRNRPGDKGENTFED

[0030] In another example, the Casl3 protein is Casl3-Cdl4, which has an amino acid sequence of SEQ ID NO: 4 as shown in Table 1:

[0031] MAKKLSPKEIREAAKAEKMKSIKAAEAEREKAAEEAKLKAEAEKKEKAEKNEREKALKRFRLDEKSRMAL PKSERKSLAKAAGVKSAFAVGNDIYLTSFDRGNDAIVEKKITDTVVTNLRSDESFEVNENTITEMSVPIKSKRISDLYAIADN PLYRKDSATKVQPDKLLLKDTLEKLYFGKTFDDTLHIQIIYNILDIEKILTVYSINTIYCLNNLFGKESGEKEDLISKLTYQITYD EFKESKAHNEFIDFYNLNTLGYYGNIFFKEKKKRSQKEIYDIIALIATIRQWCVHCEEDKRTWLFNTETVLSKEFLDILDDVY ESLVEKVNRNFLKDNKVNLQILEDVLEIKDSESREKLIRQYYRFIVTKEQKLLGFSIKKLREAMLEETEFKTDKKYDTVRSK LYKLIDFLLFTGYTTDEAEKEKALFLIKSLRESLTEETKDRIYKSEAARLWLKYENTITNKIREALNEKSISELKKDKSFDDKSI TSIITDEVSGKKATYFSKTIYLLAQFIDGKEVNDLTTSLINKFDNIRSLIDTAGQIGLDCKFTEEYKFFENSDQIRTELHVIKNL TNMEYYDTTVKKQMYKDAVHILGIQDDVSDAELEKIINSILLLNENGKPLPGTKGKKGFRNFIISNVLKSRRFIYLIKYCNPK KIRKIAGNRKIIKFVLSRITDSQLERYYYSCNPELKSGIYPGRDDAVNDLSILIADMKFEDFKNVDQSANVHDNNNAAREKM KYQTIISLYLTVCYHLVKNLVNINARYAMAFHALERDARLYQIFSSEENYVDNLNADYAILTKTLLKDNYENAGNLYLRNKK WNKLTRENLDNYIPQAAANFRNAVAHLNPIRNADMLLEDIEDVSSYYAIYHYIMQKSVTNRTIRVSNTTEDEKRILTDYQN KIKRHHGYNKDFVKALCVPFAYNIVRFKSLSIYEMFDRNYHEKTSPETDDSQSP

[0032] In another example, the Casl3 protein is Casl3-Cdl5, which has an amino acid sequence of SEQ ID NO: 6 as shown in Table 1 :

[0033] MDKEKTTVEGKNTNQKSDVLKSLAKANGLKSSFVIGNEVVMTSFGRGNSAILEKKITGSRIENLNPNVAFY VKKHISDSCNPDSGKYDVKSKRMKEKAWDDPVYVSPEKASNVHAGQDLIGCKNVLEERYFGKTFDDNIHIQLIYNILDIE KILAVHVSNATFAINNILGIEGKENEDFIGNLSVLNTFDEFENYETHPKFANKSAIKENLRKSKVFFDKIKKGNKLGYFGQAFYYATGTGKNLIFTKKSSETIYELLALVGSLRQFCVHDEVMVDNKVKSRSWLYNAQKELKPDFLKALDELYSKEVEKIDSDF IVNNTVDLHIIHDAIDVIDGSADWQKITNEYYDFIIRKCFKNIGFSIKRLRETMIEEQMKVLCGKCDRENCKGYGKCFKNKKY DSVRSRLNRIVDFIIFRHYNDEAILKNVSLLRTCMSEEEKQKRFYLPEAKALWKKYNYVFRNYVLKKLNGKSISGLKEKAIE NSIDINSVKISLGDPDYFCKFIYLLTFFLDGKEINDLLTTLINKFDNIASFISVMKNDKLSIDCEFVPEYSFFANSAQITSDLRVI NSFARMQAPAEPSKDDMYRDALDILGMDDLSEDGKKQLEDTVLCRDENGKYMKKEDNNPKRDTNFRNFLGNNVLAST RFKYLIRYNNAKKTRALANNKAVIIFMLNKINKQNPEQIVSYYKACRDDSDPVASDAEAKIEFLAEKIMNVSCTQFRYVKNG TKVRPDEAKEKERFKAIIGLYLTVMYLITKNMVYINSRYVTAFHCLERDSELHGVKFDQKKLQPNLTKKFIDPKTCGDYGL RNNKRARTYIEQNMDKMSNCTSYWNEYRNAVAHLSVIRNMNQYIKNVKNIGSCFELYHYIMQRFLLDKENIAESLREYDD FIKKKGCYRKDFVKALNTPFGYNLARYKNLSIAELFDRNDTELERTNKLRNEAIKADIEEI

[0034] The buffer as disclosed herein can not only enhance cleavage activity of the Cas 13 protein variants disclosed herein, but also can enhance other Cas protein of the same or similar clade. As used herein, the term "clade" refers to a group of organisms (or genes or proteins, in this case) that have evolved from a common ancestor. Specifically, it means that the Casl3 or Casl2 proteins as disclosed herein and any other Casl3 or Cas 12 proteins within the same clade share a common evolutionary origin and thus have similar genetic and structural characteristics.

[0035] The DNA sequence encoding SEQ ID NO: 2 is SEQ ID NO: 1. The DNA sequence encoding SEQ ID NO: 4 is SEQ ID NO: 3. The DNA sequence encoding SEQ ID NO: 6 is SEQ ID NO: 5. These DNA and amino acid sequences are shown in Table 1.

[0036] As used herein, the terms "polypeptide", "peptide", and "protein" used herein arc used interchangeably in this application to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are an artificial chemical analog of the corresponding naturally occurring amino acid, and to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence" and "protein" may also include modified forms including, but not limited to, glycosylation, lipid linkage, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. Unless otherwise stated, amino acid sequences disclosed herein are written in the N-terminal to C-terminal direction using one-letter notation. For example, when expressed as RNVP, it means a peptide in which arginine, asparagine, valine, and proline are sequentially connected from the N-terminal to the C- terminal. The method of notation of each amino acid is as follows: Alanine (Ala, A); Arginine (Arg, R); Asparagine (Asn, N); Aspartic acid (Asp, D); Cysteine (Cys, C); Glutamic acid (Glu, E); Glutamine (Gin, Q); Glycine (Gly, G); Histidine (His, H); Isoleucine (Ile, I); Leucine (Leu, L); Lysine (Lys K); Methionine (Met, M); Phenylalanine(Phe, F); Proline (Pro, P); Serine (Ser, S); Threonine (Thr, T); Tryptophan (Trp, W); Tyrosine (Tyrosine; Tyr, Y); and Valine (Vai, V).

[0037] As used herein, the terms "polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably and are single- or double-stranded RNA or DNA polymers, optionally containing synthetic unnatural, or altered nucleotide bases. Nucleotides are referred to by their single letter designations as follows: "A" for adenosine or deoxy adenosine (for RNA or DNA, respectively), "C" for cytidine or deoxycytidine, "G" for guanosine or deoxyguanosine, "U" means uridine, "T" means deoxythymidine.

[0038] The buffer as disclosed herein comprises a base composition comprising HEPES, KC1, MgCF, and glycerol. A base composition means that these components in the composition must be present in all embodiments of the buffer disclosed in this invention.

[0039] In one example, the base composition is one comprising about 20 mM HEPES, about 50 mM KC1, about 5 mM MgC12, about 5% glycerol, and having a pH of about 6.8;

[0040] In another example, the base composition is one comprising about 20 mM HEPES, about 50 mM NaCl, about 10 mM MgC12, about 5% glycerol, and having a pH of about 7.0;

[0041] In another example, the base composition is one comprising about 20 mM HEPES, about 50 mM NaCl, about 10 mM MgC12, about 5% glycerol, about I μg / ml BSA, and having a pH of about 7.0;

[0042] In another example, the base composition is one comprising about 20 mM HEPES, about 60 mM NaCl, about 6 mM MgC12, about 5% glycerol, and having a pH of about 6.8;

[0043] In another example, the base composition is one comprising about 20 mM HEPES, about 60 mM NaCl, about 5% PEG, about 5 μM DTT, and having a pH of about 6.8; and

[0044] In another example, the base composition is one comprising about 20 mM HEPES, about 60 mM KC1, about 5% PEG, and having a pH of about pH 8.0.

[0045] The buffer as disclosed herein further comprises one or more components selected from the group consisting of a salt, an organic solvent, and a protein.

[0046] The salt of the buffer as disclosed herein is selected from the group consisting of monopotassium phosphate (KH2PO4), sodium formate (HCOONa), calcium acetate (Ca(C2H3O2)2), sodium acetate (CH3COONa). ammonium sulfate ((NH4)2SO4)), potassium thiocyanate (KSCN), calcium chloride (CaCl2), sodium chloride (NaCI), Nickel(II) nitrate (Ni(NO3)2), lithium sulfate (LiSO4).

[0047] In one example, the concentration of each of the salt as disclosed herein is 5 mM-3 M, 10-60 mM, or 0.25-10 mM. In some examples, the concentration of each of the salt as disclosed herein is 0.25-10 mM, 0.25-0.5 mM, 0.5-1 mM, 1-2.5 mM, 2.5-5 mM, 5-10 mM, 0.25-9.5 mM, 1-9 mM, 1.5-8.5 mM, 2-8 mM, 3.5-7.5 mM, 4-7 mM, 4.5-6.5 mM, or 5-6 mM. In some examples, the concentration of each of the salt as disclosed herein is 10-60 mM, 10-20 mM, 20-30 mM, 30-40 mM, 40-50 mM, 50-60 mM, 20-50 mM, or 30-40 mM. In some examples, the concentration of each of the salt as disclosed herein is 5 mM-3M, 10 mM - 2 M, 20 mM - 1 M, 30 mM - 900 mM, 40 mM - 800 mM, 50 mM - 700 mM, 100-600 mM, 120 mM - 500 mM, 150 mM - 400 mM, 200 mM -300 mM, 5-15 mM, 10-20 mM, 15-30 mM, 20-40 mM, 30-50 mM, 40-100 mM, 50-200 mM, 100-300 mM, 200-400 mM, 300-500 mM, 400-600 mM, 500-700 mM, 600-800 mM, 700-900 mM, 800 mM-lM, 1-3M, or 1.5-2 M. In some examples, the concentration of each of the salt as disclosed herein is about 0.25 mM, about 0.5 mM, about 1 mM, about 2.5 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1 M, about 1.5 M, about 2 M, about 2.5 M, or about 3M.

[0048] In one example, the salt is monopotassium phosphate (KH2PO4) and the concentration of monopotassium phosphate (KH2PO4) is 10-60 mM, for example 10-30 mM, 20-40 mM, 30-50 mM, 40-60 mM, 20-50 mM, 30-40 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, or about 60 mM.

[0049] In another example, the salt is sodium formate (HCOONa) and the concentration of sodium formate (HCOONa) is 10-60 mM, for example 10-30 mM, 20- 40 mM, 30-50 mM, 40-60 mM, 20-50 mM, 30-40 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, or about 60 mM.

[0050] In another example, the salt is calcium acetate (Ca(C2H3O2)2) and the concentration of calcium acetate (Ca(C2H3O2)2) is 10-60 mM, for example 10-30 mM,20-40 mM, 30-50 mM, 40-60 mM, 20-50 mM, 30-40 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, or about 60 mM.

[0051] In another example, the salt is sodium acetate (CH3COONa) and the concentration of sodium acetate (CH3COONa) is 10-60 mM, for example 10-30 mM, 20- 40 mM, 30-50 mM, 40-60 mM, 20-50 mM, 30-40 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, or about 60 mM.

[0052] In another example, the salt is ammonium sulfate ((NH4)2SO4) and the concentration of ammonium sulfate ((NH4)2SO4) is 10-60 mM, for example 10-30 mM, 20-40 mM, 30-50 mM, 40-60 mM, 20-50 mM, 30-40 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, or about 60 mM.

[0053] hr another example, the salt is potassium thiocyanate (KSCN) and the concentration of potassium thiocyanate (KSCN) is 10-60 mM, for example 10-30 mM, 20-40 mM, 30-50 mM, 40-60 mM, 20-50 mM, 30-40 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, or about 60 mM.

[0054] In another example, the salt is calcium chloride (CaCl2) and the concentration of calcium chloride (CaCl2) is 10-60 mM, for example 10-30 mM, 20-40 mM, 30-50 mM, 40-60 mM, 20-50 mM, 30-40 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, or about 60 mM; or 1.25-50 mM, for example 1.25-10 mM, 2.5-5 mM, 5-12.5 mM, 12.5-15 mM, 10-20 mM, 15-25 mM, 20-30 mM, 25-35 mM, 30-40 mM, 35-45 mM, 40-50 mM, 2.5-45 mM, 5-40 mM, 10-35 mM, 12.5-30 mM, about 1.25 mM, about 2.5 mM, about 5 mM, about 10 mM, about 12.5 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM.

[0055] In another example, the salt is sodium chloride (NaCl) and the concentration of sodium chloride (NaCl) is 0.25-10 mM, for example 0.25-1 mM, 0.5-1.5 mM, 1-2 mM,1.5-2.5 mM, 2-3 mM, 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4-5 mM, 4.5-5.5 mM, 5-6 mM,5.5-6.5 mM, 6-7 mM, 6.5-7.5 mM, 7-8 mM, 7.5-8.5 mM, 8-9 mM, 8.5-9.5 mM, 9-10 mM, about 0.25 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM; or 1.25-50 mM, for example 1.25-10 mM, 2.5-5 mM, 5-12.5 mM, 12.5-15 mM, 10-20 mM, 15-25 mM, 20-30 mM, 25-35 mM, 30-40 mM, 35-45 mM, 40-50 mM, 2.5-45 mM, 5-40 mM, 10-35 mM, 12.5-30 mM, about1.25 mM, about 2.5 mM, about 5 mM, about 10 mM, about 12.5 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM.

[0056] In another example, the salt is Nickel(II) nitrate (Ni(NO3)2) and the concentration of Nickcl(II) nitrate (Ni(NO3)2) is 0.25-10 mM, for example 0.25-1 mM, 0.5-1.5 mM, 1-2 mM, 1.5-2.5 mM, 2-3 mM, 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4-5 mM,4.5-5.5 mM, 5-6 mM, 5.5-6.5 mM, 6-7 mM, 6.5-7.5 mM, 7-8 mM, 7.5-8.5 mM, 8-9 mM,5.5-9.5 mM, 9-10 mM, about 0.25 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM.

[0057] In another example, the salt is lithium sulfate ( LiSO4) and the concentration of lithium sulfate (LiSO4) is 0.25-10 mM, for example 0.25-1 mM, 0.5-1.5 mM, 1-2 mM,1.5-2.5 mM, 2-3 mM, 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4-5 mM, 4.5-5.5 mM, 5-6 mM,5.5-6.5 mM, 6-7 mM, 6.5-7.5 mM, 7-8 mM, 7.5-8.5 mM, 8-9 mM, 8.5-9.5 mM, 9-10 mM, about 0.25 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM.

[0058] The organic solvent of the buffer is selected from the group consisting of Tween 20, Dithiothreitol (DTT), Phenylmethylsulfonyl fluoride (PMSF), 2-Methyl-2,4- pentanediol (MPD), acetone, dimethylsulfoxide (DMSO), ethylene glycol, methanol, 2- propanol, EDTA, urea.

[0059] In one example, the concentration of each of the organic solvents disclosed herein is 5 mM - 3 M, or 1-5 mM. In some examples, the concentration of each of the organic solvents as disclosed herein is 1-5 mM, 1-2 mM, 1.5-2.5 mM, 2-3 mM, 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4-5 mM, 1.5-4.5 mM, 2-4 mM. In some examples, the concentration of each of the organic solvents as disclosed herein is 5 mM-3M, 10 mM - 2 M, 20 mM - 1 M, 30 mM - 900 mM, 40 mM - 800 mM, 50 mM - 700 mM, 100-600 mM, 120 mM - 500 mM, 150 mM - 400 mM, 200 mM -300 mM, 5-15 mM, 10-20 mM, 15-30 mM, 20-40 mM, 30-50 mM, 40-100 mM, 50-200 mM, 100-300 mM, 200-400 mM, 300-500 mM, 400-600 mM, 500-700 mM, 600-800 mM, 700-900 mM, 800 mM-lM, 1-3M, or 1.5-2 M. In some examples, the concentration of each of the organic solvents as disclosed herein is about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 100 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1 M, about 1.5 M, about 2 M, about 2.5 M, or about 3M.

[0060] In one example, the organic solvent is DTT and the concentration of DTT is 0.1-15 mM, for example 0.1-2 mM, 1-3 mM, 2-4 mM, 3-5 mM, 4-6 mM, 5-7 mM, 6-8 mM, 7-9 mM, 8-10 mM, 9-11 mM, 10-12 mM, 11-13 mM, 12-14 mM, 13-15 mM, 1-13 mM, 2-12 mM, 3-11 mM, 4-10 mM, 5-9 mM, about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM; or 1-5 mM, for example 1-2 mM, 1.5-2.5 mM, 2-3 mM, 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4-5 mM, 1-4 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM.

[0061] In another example, the organic solvent is PMSF and the concentration of PMSF is 0.1-15 mM, for example 0.1-2 mM, 1-3 mM, 2-4 mM, 3-5 mM, 4-6 mM, 5-7 mM, 6-8 mM, 7-9 mM, 8-10 mM, 9-11 mM, 10-12 mM, 11-13 mM, 12-14 mM, 13-15 mM, 1-13 mM, 2-12 mM, 3-11 mM, 4-10 mM, 5-9 mM, about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM; or 0.25-10 mM, for example 0.25-1 mM, 0.5-1.25 mM, 0.75-1.5 mM, 1-2 mM, 1.5-2.5 mM, 2-3 mM, 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4-5 mM, 4.5-5.5 mM, 5-6 mM, 5.5-6.5 mM, 6-7 mM, 6.5-7.5 mM, 7-8 mM, 7.5-8.5 mM, 8-9 mM, 8.5-9.5 mM, 9-10 mM, about 0.25 mM, about 0.5 mM, about 0.75 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM; or 1-2.5 mM, for example 1-2 mM, 1.5-2.5 mM, about 1 mM, about 1.5 mM, about 2 mM, or about2.5 mM.

[0062] In another example, the organic solvent is DMSO and the concentration of DMSO is 0.1-15 mM, for example 0.1-2 mM, 1-3 mM, 2-4 mM, 3-5 mM, 4-6 mM, 5-7mM, 6-8 mM, 7-9 mM, 8-10 mM, 9-11 mM, 10-12 mM, 11-13 mM, 12-14 mM, 13-15 mM, 1-13 mM, 2-12 mM, 3-11 mM, 4-10 mM, 5-9 mM, about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM; or 0.25-10 mM, for example 0.25-1 mM, 0.5-1.25 mM, 0.75-1.5 mM, 1-2 mM, 1.5-2.5 mM, 2-3 mM, 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4-5 mM, 4.5-5.5 mM, 5-6 mM, 5.5-6.5 mM, 6-7 mM, 6.5-7.5 mM, 7-8 mM, 7.5-8.5 mM, 8-9 mM, 8.5-9.5 mM, 9-10 mM, about 0.25 mM, about 0.5 mM, about 0.75 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM; or 1-2.5 mM, for example 1-2 mM, 1.5-2.5 mM, about 1 mM, about 1.5 mM, about 2 mM, or about 2.5 mM.

[0063] In another example, the organic solvent is Tween 20 and the concentration of Tween 20 is 0.1-15 mM, for example 0.1-2 mM, 1-3 mM, 2-4 mM, 3-5 mM, 4-6 mM, 5- 7 mM, 6-8 mM, 7-9 mM, 8-10 mM, 9-11 mM, 10-12 mM, 11-13 mM, 12-14 mM, 13-15 mM, 1-13 mM, 2-12 mM, 3-11 mM, 4-10 mM, 5-9 mM, about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM; or 0.25-10 mM, for example 0.25-1 mM, 0.5-1 .25 mM, 0.75-1 .5 mM, 1-2 mM, 1.5-2.5 mM, 2-3 mM, 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4-5 mM, 4.5-5.5 mM, 5-6 mM, 5.5-6.5 mM, 6-7 mM, 6.5-7.5 mM, 7-8 mM, 7.5-8.5 mM, 8-9 mM, 8.5-9.5 mM, 9-10 mM, about 0.25 mM, about 0.5 mM, about 0.75 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM; or about 2.5- 5 mM, for example 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4-5 mM, 2.5-4 mM, 3-3.5 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM.

[0064] In another example, the organic solvent is ethylene glycol and the concentration of ethylene glycol is 0.1-15 mM, for example 0.1-2 mM, 1-3 mM, 2-4 mM, 3-5 mM, 4-6 mM, 5-7 mM, 6-8 mM, 7-9 mM, 8-10 mM, 9-11 mM, 10-12 mM, 11-13 mM, 12-14 mM, 13-15 mM, 1-13 mM, 2-12 mM, 3-11 mM, 4-10 mM, 5-9 mM, about0.1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM; or 0.25-10 mM, for example 0.25-1 mM, 0.5-1.25 mM, 0.75-1.5 mM, 1-2 mM, 1.5-2.5 mM, 2-3 mM, 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4- 5 mM, 4.5-5.5 mM, 5-6 mM, 5.5-6.5 mM, 6-7 mM, 6.5-7.5 mM, 7-8 mM, 7.5-8.5 mM,8-9 mM, 8.5-9.5 mM, 9-10 mM, about 0.25 mM, about 0.5 mM, about 0.75 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM; or about 2.5 mM.

[0065] In another example, the organic solvent is methanol and the concentration of methanol is 0.1-10 mM, for example 0.1-2 mM, 1-3 mM, 2-4 mM, 3-5 mM, 4-6 mM, 5- 7 mM, 6-8 mM, 7-9 mM, 8-10 mM, 1-9 mM, 2-8 mM, 3-7 mM, about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM; or 0.25-5 mM, for example 0.25-1 mM, 0.5- 1.25 mM, 0.75-1.5 mM, 1-2 mM, 1.5-2.5 mM, 2-3 mM, 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4-5 mM, about 0.25 mM, about 0.5 mM, about 0.75 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM; or 1-5 mM, for example 1-3 mM, 2-4 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM.

[0066] In another example, the organic solvent is 2-propanol and the concentration of 2-propanol is 0.1-15 mM, for example 0.1-2 mM, 1-3 mM, 2-4 mM, 3-5 mM, 4-6 mM, 5-7 mM, 6-8 mM, 7-9 mM, 8-10 mM, 9-11 mM, 10-12 mM, 11-13 mM, 12-14 mM, 13- 15 mM, 1-13 mM, 2-12 mM, 3-11 mM, 4-10 mM, 5-9 mM, about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM; or 0.25-10 mM, for example 0.25-1 mM, 0.5-1.25 mM, 0.75-1.5 mM, 1-2 mM, 1.5-2.5 mM, 2-3 mM, 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4-5 mM, 4.5-5.5 mM, 5-6 mM, 5.5-6.5 mM, 6-7 mM, 6.5-7.5 mM, 7-8 mM, 7.5-8.5 mM, 8-9 mM, 8.5-9.5 mM,9-10 mM, about 0.25 mM, about 0.5 mM, about 0.75 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM,about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM; or 1-2.5 mM, for example 1-2 mM, 1.5-2.5 mM, about 1 mM, about 1.5 mM, about 2 mM, or about2.5 mM.

[0067] In another example, the organic solvent is acetone and the concentration of acetone is 0.1-15 mM, for example 0.1-2 mM, 1-3 mM, 2-4 mM, 3-5 mM, 4-6 mM, 5-7 mM, 6-8 mM, 7-9 mM, 8-10 mM, 9-11 mM, 10-12 mM, 11-13 mM, 12-14 mM, 13-15 mM, 1-13 mM, 2-12 mM, 3-11 mM, 4-10 mM, 5-9 mM, about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM; or 0.25-10 mM, for example 0.25-1 mM, 0.5-1.25 mM, 0.75-1.5 mM, 1-2 mM, 1.5-2.5 mM, 2-3 mM, 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4-5 mM, 4.5-5.5 mM, 5-6 mM, 5.5-6.5 mM, 6-7 mM, 6.5-7.5 mM, 7-8 mM, 7.5-8.5 mM, 8-9 mM, 8.5-9.5 mM, 9-10 mM, about 0.25 mM, about 0.5 mM, about 0.75 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM; or 1-2.5 mM, for example 1-2 mM, 1.5-2.5 mM, about 1 mM, about 1.5 mM, about 2 mM, or about2.5 mM.

[0068] In another example, the organic solvent is MDP and the concentration of MDP is 0.1-15 mM, for example 0.1-2 mM, 1-3 mM, 2-4 mM, 3-5 mM, 4-6 mM, 5-7 mM, 6-8 mM, 7-9 mM, 8-10 mM, 9-11 mM, 10-12 mM, 11-13 mM, 12-14 mM, 13-15 mM, 1-13 mM, 2-12 mM, 3-11 mM, 4-10 mM, 5-9 mM, about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM; or 0.25-10 mM, for example 0.25-1 mM, 0.5-1.25 mM, 0.75-1.5 mM, 1-2 mM, 1.5-2.5 mM, 2-3 mM, 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4-5 mM, 4.5-5.5 mM, 5-6 mM, 5.5-6.5 mM, 6-7 mM, 6.5-7.5 mM, 7-8 mM, 7.5-8.5 mM, 8-9 mM, 8.5-9.5 mM, 9-10 mM, about 0.25 mM, about 0.5 mM, about 0.75 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM; or about 4 mM.

[0069] In another example, the organic solvent is EDTA and the concentration of EDTA is 0.1-5 mM, for example 0.1-2 mM, 1-3 mM, 2-4 mM, 3-5 mM, 0.5-4 mM, 1-3 mM, about 0.1 mM, about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM; or 0.25-1 mM, for example 0.25-0.5 mM, 0.5-0.75 mM, 0.75-1 mM, about 0.25 mM, about 0.5 mM, about 0.75 mM, or about 1 mM.

[0070] In another example, the organic solvent is urea and the concentration of urea is 0.1-15 mM, for example 0.1-2 mM, 1-3 mM, 2-4 mM, 3-5 mM, 4-6 mM, 5-7 mM, 6-8 mM, 7-9 mM, 8-10 mM, 9-11 mM, 10-12 mM, 11-13 mM, 12-14 mM, 13-15 mM, 1-13 mM, 2-12 mM, 3-11 mM, 4-10 mM, 5-9 mM, about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM; or 0.25-10 mM, for example 0.25-1 mM, 0.5-1.25 mM, 0.75-1.5 mM, 1-2 mM, 1.5-2.5 mM, 2-3 mM, 2.5-3.5 mM, 3-4 mM, 3.5-4.5 mM, 4-5 mM, 4.5-5.5 mM, 5-6 mM, 5.5-6.5 mM, 6-7 mM, 6.5-7.5 mM, 7-8 mM, 7.5-8.5 mM, 8-9 mM, 8.5-9.5 mM, 9-10 mM, about 0.25 mM, about 0.5 mM, about 0.75 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, or about 10 mM.

[0071] The protein of the buffer is casein.

[0072] In one example, the concentration of casein disclosed herein is 0.001-10 mg / mL, or 0.01-1 mg / mL. In some examples, the concentration of each of the proteins as disclosed herein is 0.001-0.005 mg / mL, 0.0015-0.003 mg / mL, 0.003-0.006 mg / mL, 0.005-0.01 mg / mL, 0.006-0.015 mg / mL, 0.01-0.03 mg / mL, 0.015-0.03 mg / mL, 0.03- 0.05 mg / mL, 0.03-0.06 mg / mL, 0.04-0.06 mg / mL, 0.05-0.07 mg / mL, 0.06-0.08 mg / mL, 0.07-0.09 mg / mL, 0.08-0.1 mg / mL, 0.1-0.3 mg / mL, 0.2-0.4 mg / mL, 0.3-0.5 mg / mL, 0.4- 0.6 mg / mL, 0.5-0.7 mg / mL, 0.6-0.8 mg / mL, 0.7-0.9 mg / mL, 0.8-1 mg / mL, 1-2 mg / mL, 2-3 mg / mL, 3-4 mg / mL, 4-5 mg / mL, 5-6 mg / mL, 6-7 mg / mL, 7-8 mg / mL, 8-9 mg / mL, 9-10 mg / mL, about 0.001 mg / mL, about 0.005 mg / mL, about 0.01 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1 mg / mL, about 2 mg / mL, about2.5 mg / mL, about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, about 5 mg / mL, about 5.5 mg / mL, about 6 mg / mL, about 6.5 mg / mL, about 7 mg / mL, about7.5 mg / mL, about 8 mg / mL, about 8.5 mg / mL, about 9 mg / mL, about 9.5 mg / mL, or about 10 mg / mL.

[0073] In another example, the concentration of casein is 0.005-0.05 mg / mL, or 0.0015-0.06 mg / mL, for example 0.0015-0.003 mg / mL, 0.003-0.006 mg / mL, 0.005-0.01 mg / mL, 0.006-0.015 mg / mL, 0.015-0.03 mg / mL, 0.03-0.06 mg / mL, 0.04-0.05 mg / mL, 0.01-0.03 mg / mL, 0.02-0.04 mg / mL, 0.03-0.05 mg / mL, about 0.001 mg / mL, about 0.0015 mg / mL, about 0.005 mg / mL, about 0.1 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, or about 0.06 mg / mL.

[0074] The buffer as disclosed herein has a PH of between 6.0 and 8.0. In one example, the buffer as disclosed herein has a PH of about 6.0. In another example, the buffer as disclosed herein has a PH of about 6.5. In another example, the buffer as disclosed herein has a PH of about 7.0. In another example, the buffer as disclosed herein has a PH of about 7.5. In another example, the buffer as disclosed herein has a PH of about 8.0.

[0075] The buffer as disclosed herein possess unique advantages of enhancing the enzymatic cleavage activity of a Cas protein as disclosed herein by 1.5-20 fond, for example 1.76-3.04 fold, 1.88-2.70 fold, or 10.4-11.2 fold; for example 1.5-2 fold, 1.76-2.5 fold, 2-4 fold, 3-5 fold, 4-6 fold, 5-7 fold, 7-9 fold, 8-10 fold, 9-11 fold, about 1.2 fold, about 1 .5 fold, about 1 .76 fold, about 1 .88 fold, about 1 .9 fold, about 2.03 fold, about 2.21 fold, about 2.24 fold, about 2.6 fold, about 2.67 fold, about 2.70 fold, about 3 fold, about 3.04 fold, about 3.9 fold, about 4 fold, about 5 fold, about 5.5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 10.4 fold, about 11 fold, about 11.2 fold, about 12 fold, about 13 fold, about 14 fold, about 15 fold, about 16 fold, about 17 fold, about 18 fold, about 19 fold, about 20 fold, compared to the enzymatic cleavage activity of a Cas protein in the absence of the buffer as disclosed herein.

[0076] In one example, the buffer comprises the base composition as disclosed herein, and one or more salts as disclosed herein. In another example, the buffer comprises the base composition as disclosed herein, and one salt as disclosed herein. In another example, the buffer comprises the base composition as disclosed herein, and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 salts as disclosed herein.

[0077] In another example, the buffer comprises the base composition as disclosed herein, and one or more organic solvents disclosed herein. In another example, the buffer comprises the base composition as disclosed herein, and one organic solvent as disclosed herein. In another example, the buffer comprises the base composition as disclosed herein, and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 organic solvents as disclosed herein.

[0078] In another example, the buffer comprises the base composition as disclosed herein, and the protein disclosed herein which is casein.

[0079] In another example, the buffer comprises the base composition as disclosed herein, one or more salts (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 salts) as disclosed herein, and one or more organic solvents (such as 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, or 15 organic solvents) as disclosed herein.

[0080] In another example, the buffer comprises the base composition as disclosed herein, one or more salts (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 salts) as disclosed herein, and the protein which is casein as disclosed herein.

[0081] In another example, the buffer comprises the base composition as disclosed herein, one or more organic solvents (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 organic solvents) as disclosed herein, and the protein which is casein as disclosed herein.

[0082] In another example, the buffer comprises the base composition as disclosed herein, one or more salts (such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, or 17 salts) as disclosed herein, one or more organic solvents (such as 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 organic solvents) as disclosed herein, and the protein such as casein as disclosed herein.

[0083] In another aspect, the present disclosure refers to a detection kit for a nucleic acid, comprising:(a) the buffer as disclosed herein;(b) a Cas protein selected from the group consisting of Casl3d, Casl3a, Casl3b, Casl3c, Casl2a, Casl2b, Casl2c, Casl2d, and Casl2f; and(c) a crRNA configured to target the nucleic acid and form a complex with the Cas protein.

[0084] As used herein, "kit" means a collection of at least one component constituting the kit. Together, the components constitute a functional unit for a given purpose.Individual member components may be physically packaged together or separately. For example, a kit comprising an i nst ruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory' device or downloaded from an internet website, or as recorded presentation.

[0085] The detection kit comprises the buffer as disclosed herein which can enhance the enzymatic cleavage activity of a Cas protein selected from the group consisting of Casl3d, Casl3a, Casl3b, Casl3c, Casl2a, Casl2b, Casl2c, Casl2d, and Casl2f, a Cas protein disclosed herein, and a crRNA configured to target the nucleic acid and form a complex with the Cas protein.

[0086] As used herein, the term "CRISPR RNA (crRNA)" refers to a crucial component of the CRISPR-Cas system. crRNA is transcribed from the CRISPR locus, which contains a series of short, repetitive DNA sequences (direct repeat) interspaced with unique sequences called protospacers or spacers. These spacers are derived from previous infections by viruses or other mobile genetic elements. crRNA consists of a repeat-derived sequence (direct repeat) and a spacer sequence (protospaccr). The repeat- derived sequence is conserved, while the spacer sequence is complementary to the target DNA sequence. The degree of complementarity between a crRNA and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). The primary role of crRNA is to guide the Cas nuclease (such as Cas 13 or Cas 12) to the target nucleic acid such as DNA or RNA during the interference step of the CRISPR-Cas immune response. The crRNA forms a complex with the Cas protein (such as Casl3 or Casl2), and this complex then binds to the target nucleic acid DNA or RNA sequence, leading to its cleavage and subsequent degradation.

[0087] The crRNA is configured to target the nucleic acid and form a complex with the Cas protein. As used herein, the term "target" means “binding”, which can refer to non- covalent physical association of a first and a second moiety (such as a crRNA and a target sequence, or a CRISPR-Cas complex and a target sequence) wherein the association between the first and second moictics is at least 2 times as strong, at least 5 times as strong as, at least 10 times as strong as, at least 50 times as strong as, at least 100 times as strong as, or stronger than the association of either moiety with most or all other moieties present in the environment in which binding occurs. Binding of two or more entities may be considered specific if the equilibrium dissociation constant, Kd, is 10"3M or less, 10"4M or less, 10'5M or less, 10'6M or less, 10"7M or less, 10"8M or less, 10"9M or less, 1010M or less, 1011M or less, or 1012M or less under the conditions employed, e.g., under physiological conditions such as those inside a cell or consistent with cell survival. In some embodiments, specific binding can be accomplished by a plurality of weaker interactions (e.g., a plurality of individual interactions, wherein each individual interaction is characterized by a Kd of greater than 10"3M).

[0088] The term "complex" refers to the complex formed by the combination of CRISPR RNA (crRNA) and the Cas 12 or Cas 13 protein as disclosed herein, i.c. a ribonucleoprotein complex. The crRNA can hybridize with the target sequence and thus the complex recognizes and cleaves polynucleotides that hybridize to the crRNA.

[0089] In one example, the Cas protein and the crRNA are lyophilized for easy transportation and storage, and can be reconstituted by water or a suitable buffer upon usage.

[0090] Thus, in the context of the formation of a CRISPR-Casl2 or CRISPR-Casl3 complex, "target sequence" refers to a polynucleotide such as DNA or RNA targeted by a crRNA, e.g., a polynucleotide sequence complementary to the crRNA, wherein the hybridization between the target sequence and the crRNA will promote Cas protein to exert its activity, such as the activity of cutting the target sequence. Full complementarity is not required, as long as there is sufficient complementarity to cause hybridization and facilitate Cas protein to exert its activity. A target sequence can include any polynucleotide, such as DNA or RNA. In some examples, the target sequence is located in the nucleus or cytoplasm of a cell. In some examples, the target sequence may be located in an organelle of a eukaryotic cell such as the mitochondria or chloroplast. Insome examples, the target sequence can be a sequence encoding a gene product (e.g. protein) or a non-coding sequence (e.g. regulatory polynucleotide or dummy DNA). In one example, the nucleic acid to be detected by the detection kit as disclosed herein is DNA. In another example, the nucleic acid to be detected by the detection kit as disclosed herein is RNA.

[0091] In another example, the detection kit may further comprise a detecting agent comprising a probe nucleic acid, wherein the detecting agent generates a detectable signal when the probe nucleic acid is cleaved by the Cas protein such as a Cas 12 or Cas 13 protein as disclosed herein. In one example, the probe nucleic acid is a fluorescent probe, wherein the 5' end of the fluorescent probe sequence is labeled with a fluorescent group, and the 3' end of the fluorescent probe sequence is labeled with a quenching group. In some examples, the fluorescent group is selected from the group consisting of FAM, VIC, HEX, TRT, Cy3, Cy5, ROX, IOE and Texas Red, and the quenching group is selected from the group consisting of IABKFQ, DABCYL, MGB, BHQ-1, BHQ-2 and BHQ-3. The effect of the fluorescent probe in the CR1SPR detection system is that after the probe is nonspecifically cut by Cas protein, the fluorescent group and the quenching group are separated, the quenching group removes the blocking effect on the fluorescent group, and the change of fluorescence intensity can be detected by means of a fluorescent signal detection device under the condition of light irradiation with specific wavelength. In another example, / 56-FAM / rUrUrUrUrU / 3IABkFQ / is used as a fluorescent probe to detect the enzymatic cleavage activity of Cas 13. A fluorescence signal generated upon cutting of a fluorescent probe can be detected by means of a fluorescence detection equipment such as a fluorescence spectrometer, fluorometer, fluorescence microscopes, microplate reader, real-time PCR, flow cytometry, and fluorescence scanners.

[0092] hi another example, the probe nucleic acid is a colorimetric probe. In some examples, the colorimetric probe comprises FAM and Biotin. In another example, the colorimetric probe can be detected by a lateral flow strip. Lateral flow strips use gold nanoparticles or colored latex beads conjugated with detection molecules. Cleavage events by CRISPR-Cas lead to a visible color change on the strip. In another example, / 56-FAM / CCCCCCCC / 3Biotin / is used as a colorimetric probe to detect the enzymatic cleavage activity of Cas 12a. In another example, the colorimetric probe used to detect the enzymatic cleavage activity of Casl3 is selected from the group consisting of / 56-FAM / rUrUrUrUrU / 3Bio / , / 56-FAM / rUrUrUrUrU / 3DIG / , / 56-FAM / rArArArArA / 3Bio / , and / 56-FAM / rArArArArA / 3DIG / . A colorimetric signal can be directly visually detected by naked eyes without an instrument, or with a colorimetric reader such as a colorimeter, spectrophotometer, color analyzer, chromameter, etc.

[0093] In another example, the probe nucleic acid has a concentration of 100-3200 nM, for example about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 800 nM, about 1000 nM, about 1200 nM, about 1600 nM, about 2000 nM, about 2400 nM, about 2800 nM, about 3000 nM, or about 3200 nM.

[0094] In another example, CRISPR-Cas cleavage events can be transduced into an electrochemical signal using modified electrodes or nanoparticle-based sensors.

[0095] The shelf-life of the Cas protein and the crRNA in the detection kit is increased by 30-300% in the presence of the buffer as disclosed herein, compared to the self-life of the Cas protein and the crRNA in the absence of the buffer as disclosed herein. In some examples, in the presence of the buffer as disclosed herein, the shelf-life of the Cas protein and the crRNA in the detection kit is increased by 30-100%, 50-150%, 100-200%, 150- 250%, 200-300%, compared to the self-life of the Cas protein and the crRNA in the absence of the buffer as disclosed herein.

[0096] hr another example, the detection kit further comprises nucleic acid amplification primers, wherein the nucleic acid amplification primers arc PCR, RPA or LAMP primer pairs.

[0097] The disclosed CRISPR-Cas based detection system is compatible with lateral flow strips for visual detection of the detectable signal, making it highly suitable for point- of-care testing applications. This combination of high sensitivity, specificity, and ease of use makes the method particularly valuable for detecting low-abundance nucleic acids in diagnostics and other applications.

[0098] In another example, the Cas protein, such as Casl2 or Casl3 protein and the crRNA are in the form of a ribonucleoprotein complex. In another example, the concentration of the Cas protein used to form ribonucleoprotein complex is 200-3200 nM, for example, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM, about 1000 nM, about 1050 nM, about 1100 nM, about 1150 nM, about 1200 nM, about 1250nM, about 1300 nM, about 1350 nM, about 1400 nM, about 1450 nM, about 1500 nM, about 1550 nM, about 1600 nM, about 1650 nM, about 1700 nM, about 1750 nM, about 1800 nM, about 1850 nM, about 1900 nM, about 1950 nM, about 2000 nM, about 2050 nM, about 2100 nM, about 2150 nM, about 2200 nM, about 2250 nM, about 2300 nM, about 2350 nM, about 2400 nM, about 2450 nM, about 2500 nM, about 2550 nM, about 2600 nM, about 2650 nM, about 2700 nM, about 2750 nM, about 2800 nM, about 2850 nM, about 2900 nM, about 2950 nM, about 3000 nM, about 3050 nM, about 3100 nM, about 3150 nM, or about 3200 nM. In another example, the concentration of crRNA used to form ribonucleoprotein complex is 200-3200 nM, for example, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM, about 1000 nM, about 1050 nM, about 1100 nM, about 1150 nM, about 1200 nM, about 1250 nM, about 1300 nM, about 1350 nM, about 1400 nM, about 1450 nM, about 1500 nM, about 1550 nM, about 1600 nM, about 1650 nM, about 1700 nM, about 1750 nM, about 1800 nM, about 1850 nM, about 1900 nM, about 1950 nM, about 2000 nM, about 2050 nM, about 2100 nM, about 2150 nM, about 2200 nM, about 2250 nM, about 2300 nM, about 2350 nM, about 2400 nM, about 2450 nM, about 2500 nM, about 2550 nM, about 2600 nM, about 2650 nM, about 2700 nM, about 2750 nM, about 2800 nM, about 2850 nM, about 2900 nM, about 2950 nM, about 3000 nM, about 3050 nM, about 3100 nM, about 3150 nM, or about 3200 nM.

[0099] In another aspect, the present disclosure refers to a method of detecting a nucleic acid in a sample by clustered regularly interspaced short palindromic repeats and CRISPR associated protein (CRISPR-cas), using the detection kit as disclosed herein, the method comprising: incubating the sample containing the nucleic acid with the Cas protein and the crRNA, wherein when the nucleic acid is present in the sample, enzymatic cleavage activity of the Cas protein is induced to generate a detectable signal when the probe nucleic acid is cleaved; wherein the buffer enhances the enzymatic cleavage activity of the Cas protein.

[0100] As used herein, enzymatic cleavage activity of a Cas protein refers to its ability to indiscriminately cleave single-stranded nucleic acids in the vicinity once it has been activated by recognizing and binding to a specific target nucleic acid sequence. This activity is also known as trans-cleavage activity.

[0101] In one example, the nucleic acid in the sample can be amplified using a technology selected from the group consisting of Polymerase Chain Reaction (PCR), Recombinase Polymerase Amplification (RPA), Loop-Mediated Isothermal Amplification (LAMP), and Rolling Circle Amplification (RCA) prior to carrying out the detection method as disclosed herein. In another example, the method of detecting a nucleic acid in a sample as disclosed herein does not comprise pre-amplifying the nucleic acid in the sample.

[0102] In another example, the method of detecting a nucleic acid in a sample further comprising using a detection method selected from the group consisting of a colorimetric detection method, a fluorescent detection method and an electrochemical detection method to detect the detectable signal which is a colorimetric signal, a fluorescent signal, or an electrochemical signal. In another example, the colorimetric detection method uses a lateral flow strip and a colorimetric probe comprising FAM and Biotin. In another example, the fluorescent detection method uses a fluorescent probe, wherein the fluorescence signal is measured using a fluorometer, a microplate reader, or Real-Time PCR. In another example, the electrochemical detection method transduces CRISPR-Cas cleavage event to an electrochemical signal using a modified electrode or a nanoparticlc- based sensor.

[0103] In one example, the Cas protein used in the detection kit to be used in the method of detecting a nucleic acid in a sample as disclosed herein, is a Casl 2 protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d, and Casl2f. In another example, the Cas protein used in the detection kit to be used in the method of detecting a nucleic acid in a sample as disclosed herein, is a Cas 13 protein selected from the group consisting of Casl3d, Casl3a, Casl3b, Casl3c. In another example, the Casl3d protein is selected from the group consisting of Casl3-Cdl3 having an amino acid sequence of SEQ ID NO: 2, Casl3-Cdl4 having an amino acid sequence of SEQ ID NO: 4, and Casl3-Cdl5 having an amino acid sequence of SEQ ID NO: 6.

[0104] In one example, the method of detecting a nucleic acid in a sample as disclosed herein is performed at a temperature of 20-60 °C. In another example, the method of detecting a nucleic acid in a sample as disclosed herein is performed at a temperature selected from the group consisting of 20-25 °C, 25-30 °C, 30-35 °C, 35-40 °C, 40-45 °C, 45-50 °C, 50-55 °C, 55-60 °C, about 20 °C, about 25 °C, about 30 °C, about 35 °C, about37 C, about 40 C, about 45 C, about 50 C, about 55 °C, and about 60 C. In one particular example, the method of detecting a nucleic acid in a sample as disclosed herein is performed at a temperature of about 37 °C.

[0105] hr one example, the nucleic acid to be detected by the detection method as disclosed herein is DNA and the DNA is 10 copics / μL or less, for example, as low as about 0.0015 copies / μL. In another example, the DNA to be detected method as disclosed herein is 0.0015-0.01 copies / μL, 0.0075-0.015 copies / μL, 0.01-0.015 copies / μL, 0.015- 0.15 copies / μL, 0.15-1.5 copies / μL, 1-3 copies / μL, 1.5-3.5 copies / μL, 2-4 copies / μL, 2.5- 4.5 copies / μL, 3-5 copies / μL, 3.5-5.5 copies / μL, 4-6 copies / μL, 4.5-6.5 copies / μL, 5-7 copies / μL, 5.5-7.5 copies / μL, 6-8 copies / μL, 6.5-8.5 copies / μL, 7-9 copies / μL, 7.5-9.5 copies / μL, 8-10 copies / μL, less than 0.0015 copies / μL, less than 0.0075 copies / μL, less than 0.01 copies / μL, less than 0.015 copies / μL, less than 0.1 copies / μL, less than 0.15 copies / μL, less than 1 copies / μL, less than 1.5 copies / μL, less than 2 copies / μL, less than 3 copies / μL, less than 5 copies / μL, less than 6 copies / μL, less than 10 copies / μL, less than 15 copies / μL, less than 20 copies / μL, less than 30 copies / μL, less than 40 copies / μL, less than 50 copies / μL, less than 60 copies / μL, less than 70 copies / μL, less than 80 copics / μL, less than 90 copics / μL, less than 100 copics / μL, or less than 150 copics / μL. In another example, the DNA to be detected by the detected method as disclosed herein is about 0.0015 copics / μL, about 0.0075 copics / μL, about 0.01 copics / μL, about 0.015 copies / μL, about 0.1 copies / μL, about 0.15 copies / μL, about 0.2 copies / μL, about 0.3 copies / μL, about 0.4 copies / μL, about 0.5 copies / μL, about 0.6 copies / μL, about 0.7 copies / μL, about 0.8 copies / μL, about 0.9 copies / μL, about 1 copies / μL, about 1.5 copies / μL, about 2 copies / μL, about 2.5 copies / μL, about 3 copies / μL, about 3.5 copies / μL, about 4 copies / μL, about 4.5 copies / μL, about 5 copies / μL, about 5.5 copies / μL, about 6 copies / μL, about 6.5 copies / μL, about 7 copies / μL, about 7.5 copies / μL, about 8 copies / μL, about 8.5 copies / μL, about 9 copies / μL, about 9.5 copies / μL, about 10 copies / μL, about 11 copies / μL, about 12 copies / μL, about 13 copies / μL, about 14 copies / μL, about 15 copies / μL, about 16 copies / μL, about 17 copies / μL, about 18 copies / μL, about 19 copies / μL, about 20 copies / μL, about 30 copies / μL, about 40 copies / μL, about 50 copies / μL, about 60 copies / μL, about 70 copics / μL, about 80 copics / μL, about 90 copics / μL, about 100 copics / μL, about 110 copies / μL, about 120 copies / μL, about 130 copies / μL, about 140 copies / μL, about 150copics / uL, or more. In another example, no DNA amplification is required before detection using the disclosed detection method.

[0106] Advantageously, the present disclosure showcases superior detection of low concentrations of DNA using the present amplification-free CRISPR-Cas-based detection system with the aid of the buffer as disclosed herein, which significantly enhanced the enzymatic cleavage activity of the Cas protein such as Casl2 or Casl3.

[0107] In another example, the sample comprising the nuclei acid is a biological sample obtained from a tissue sample, saliva, blood, plasma, milk, sera, stool, urine, sputum, mucous, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, nasal swab specimen, or swab of skin or a mucosal membrane surface or a combination thereof. In another example, the biological sample is a crude sample. In another example, the nucleic acid is not purified or amplified from the biological sample.

[0108] In another example, the sample comprising the nuclei acid is a chemical sample is obtained from a pharmaceutical substance, a chemical reagent, a cosmetic product, an industrial effluent, a pollutant, a hazardous material, a polymer, an adhesive, a solvent, an oil, a gas, a metal alloy, a nanomaterial, a fuel, a cleaning agent, a dye, or a combination thereof. In another example, the chemical sample is a crude sample. In another example, the nucleic acid is not purified or amplified from the chemical sample.

[0109] In another example, the sample comprising the nuclei acid is an environmental sample is obtained from a food, a beverage, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a waste water sample, a saline water sample, exposure to atmospheric air or other gas sample, or a combination thereof. In another example, the environmental sample is a crude sample. In another example, the nucleic acid is not purified or amplified from the environmental sample.

[0110] In another example, the nucleic acid in the sample belongs to a microbe selected from a bacterium, a virus, an archaea, a protozoa, a fungi, an algae, a slime mode, a lichen, and a prion. In another example, the nucleic acid in the sample belongs to a plant, an animal, an insect, a microbe as disclosed herein, or a combination thereof.

[0111] In another example, the virus is selected from a group consisting of a doublestranded DNA virus, a single-stranded DNA virus, a double-stranded RNA virus, a positive sense RNA virus, a negative sense RNA virus, and a retrovirus.

[0112] In another example, the virus is selected from a group consisting of a coronavirus, an Ebola virus, measles, SARS, Chikungunya virus, Marburg, MERS, Dengue, Lassa, influenza, rhabdovirus, HIV, a hepatitis virus (including hepatitis A, B, C, D, or E), an influenza virus (including an influenza A or influenza B), a human respiratory syncytial virus, Sudan cbola virus, Bundibugyo virus, Tai Forest cbola virus, Reston ebola virus, Achimota virus, Aedes flavivirus, Aguacate virus, Akabane virus, Alethinophid reptarenavirus, Allpahuayo mammarenavirus, Amapari mmarenavirus, Andes virus, Apoi virus, Aravan virus, Aroa virus, Arumwot virus, Atlantic salmon paramyxovirus, Australian bat lyssavirus, Avian bomavirus, Avian metapneumovirus, Avian paramyxoviruses, penguin or Falkland Islandsvirus, BK polyomavirus, Bagaza virus, Banna virus, Bat herpesvirus, Bat sapovirus, Bear Canon mammarenavirus, Beilong virus, Betacoronavirus, Betapapillomavirus 1-6, Bhanja virus, Bokeloh bat lyssavirus, Boma disease virus, Bourbon virus, Bovine hepacivirus, Bovine parainfluenza virus 3, Bovine respiratory syncytial virus, Brazoran virus, Bunyamwera virus, Caliciviridae virus. California encephalitis virus, Candiru virus, Canine distemper virus, Canine pneumovirus, Cedar virus, Cell fusing agent virus, Cetacean morbillivirus, Chandipura virus, Chaoyang virus, Chaparc mammarenavirus, Colobus monkey papillomavirus, Colorado tick fever virus, Cowpox virus, Crimean-Congo hemorrhagic fever virus, Culcx flavivirus, Cupixi mammarenavirus, Dengue virus, Dobrava-Bclgradc virus, Donggang virus, Dugbe virus, Duvenhage virus, Eastern equine encephalitis virus, Entebbe bat virus, Enterovirus A-D, European bat lyssavirus 1-2, Eyach virus, Feline morbillivirus, Fer-de- Lance paramyxovirus, Fitzroy River virus, Flaviviridae virus, Flexal mammarenavirus, GB virus C, Gairo virus, Gemycircularvirus, Goose paramyxovirus SF02, Great Island virus, Guanarito mammarenavirus, Hantaan virus, Hantavirus Z10, Heartland virus, Hendra virus, Hepatitis A / B / C / E, Hepatitis delta virus, Human bocavirus, Human coronavirus, Human endogenous retrovirus K, Human enteric coronavirus, Human genital-associated circular DNA virus- 1, Human herpesvirus 1-8, Human mastadenovirus A-G, Human papillomavirus, Human parainfluenza virus 1-4, Human paraechovirus, Human picomavirus, Human smacovirus, Ikoma lyssavirus, Hheus virus, Influenza A-C, Ippy mammarenavirus, Irkut virus, J-virus, JC polyomavirus, Japanese encephalitis virus, Junin mammarenavirus, KI polyomavirus, Kadipiro virus, Kamiti River virus, Kedougou virus, Khujand virus, Kokobera virus, Kyasanur forestdisease virus, Lagos bat virus, Langat virus, Lassa mammarenavirus, Latino mammarenavirus, Leopards Hill virus, Liao ning virus, Ljungan virus, Lloviu virus, Louping ill virus, Lujo mammarenavirus, Luna mammarenavirus, Lunk virus, Lymphocytic choriomeningitis mammarenavirus, Lyssavirus Ozemoe, MSSI2Y225 virus, Machupo mammarenavirus, Mamastrovirus 1, Manzanilla virus, Mapucra virus, Marburg virus, Mayaro virus, Measles virus, Menangle virus, Mercadeo virus, Merkel cell polyomavirus, Middle East respiratory syndrome coronavirus, Mobala mammarenavirus, Modoc virus, Moijang virus, Mokolo virus, Monkeypox virus, Montana myotis leukoenchalitis virus, Mopeia lassa virus reassortant 29, Mopeia mammarenavirus, Morogoro virus, Mossman virus, Mumps virus, Murine pneumonia virus, Murray Valley encephalitis virus, Nariva virus, Newcastle disease virus, Nipah virus, Norwalk virus, Norway rat hepacivirus, Ntaya virus, O'nyong-nyong virus, Oliveros mammarenavirus, Omsk hemorrhagic fever virus, Oropouche virus, Parainfluenza virus 5, Parana mammarenavirus, Parramatta River virus, Peste-des-petits- ruminants virus, Pichande mammarenavirus, Picomaviridae virus, Pirital mammarenavirus, Piscihepevirus A, Porcine parainfluenza virus 1, porcine rubulavirus, Powassan virus, Primate T-lymphotropic virus 1-2, Primate cry throparvo virus 1, Punta Toro virus, Puumala virus, Quang Binh virus, Rabies virus, Razdan virus, Reptile bomavirus 1, Rhinovirus A-B, Rift Valley fever virus, Rinderpest virus, Rio Bravo virus, Rodent Torque Teno virus, Rodent hepacivirus, Ross River virus, Rotavirus A-T, Royal Farm virus, Rubella virus, Sabia mammarenavirus, Salem virus. Sandfly fever Naples virus, Sandfly fever Sicilian virus, Sapporo virus, Sathuperi virus, Seal anellovirus, Semliki Forest virus, Sendai virus, Seoul virus, Sepik virus, Severe acute respiratory syndrome-related coronavirus, Severe fever with thrombocytopenia syndrome virus, Shamonda virus, Shimoni bat virus, Shuni virus, Simbu virus, Simian torque teno virus, Simian virus 40-41, Sin Nombre virus, Sindbis virus, Small anellovirus, Sosuga virus, Spanish goat encephalitis virus, Spondweni virus, St. Louis encephalitis virus, Sunshine virus, TTV-like mini virus, Tacaribe mammarenavirus, Taila virus, Tamana bat virus, Tamiami mammarenavirus, Tembusu virus, Thogoto virus, Thottapalayam virus, Tick- bome encephalitis vims, Tioman virus, Togaviridae virus, Torque teno canis virus, Torque teno douroucouli virus, Torque teno fclis virus, Torque teno midi virus, Torque teno sus virus, Torque teno tamarin virus, Torque teno virus, Torque teno zalophus virus,Tuhoko virus, Tula virus, Tupaia paramyxovirus, Usutu virus, Uukuniemi virus, Vaccinia virus. Variola virus, Venezuelan Vesicular- stomatitis Indiana virus, WU Polyomavirus, Wesselsbron virus, West Caucasian bat virus, West Nile virus, Western equine encephalitis virus, Whitewater Arroyo mammarenavirus, Yellow fever virus, Yokose virus, Yug Bogdanovac virus, Zaire ebolavirus, Zika virus, or Zygosaccharomyccs bailii virus Z viral sequence, Canine Parvovirus, Canine Distemper Virus, Canine Coronavirus, Canine Influenza Virus, Feline Herpesvirus, Feline Calicivirus, Feline Leukemia Virus, Feline Immunodeficiency Virus, Canine Adenovirus - Type 1 and Type 2, Canine Herpesvirus, Tobacco Mosaic Virus, Potato Virus Y, Tomato Spotted Wilt Virus, Cucumber Mosaic Virus, Potato Virus X, Barley Yellow Dwarf Virus, Bean Common Mosaic Virus, Maize Dwarf Mosaic Virus, Soybean Mosaic Virus, Citrus Tristeza Virus, Rice Yellow Mottle Virus, Plum Pox Virus, Turnip Mosaic Virus, Aphid-Transmitted Yellow Dwarf Viruses, Cotton Leaf Curl Virus, Papaya Ringspot Virus, Cassava Mosaic Virus, Rice Tungro Virus, Apple Mosaic Virus, Sugarcane Mosaic Virus or a combination thereof. In a particular example, the coronavirus is SARS-CoV-2. In another particular example, the nucleic acid in the sample is a SARS-CoV-2 N gene or a fragment thereof. In another example, the nucleic acid in the sample is a SARS-CoV-2 N gene or a fragment thereof and comprise a sequence of SEQ ID NO: 14.

[0113] In another example, the bacteria is a Gram-positive bacterium or a Gramnegative bacterium selected from the group consisting of Staphylococcus aureus, Staphylococcus epide midis, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile and Clostridium tetani, Corynebacterium diphtheria, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Chlamydia trachomatis, Yersinia pestis, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, and Vibrio cholera, Brucella abortus, Brucella melitensis, Brucella suis, Brucella canis, Brucella ovis, Brucella neotomae, Mycoplasma mycoides, Clostridium perfringens, Mycobacterium avium Salmonella spp., Pasteurella multocida, Haemophilus parasuis, Actinobacilluspleuropneumoniae, Yersinia pestis, Francisella tularensis, Bacillus anthracis, Coxiella burnetiid, Listeria monocytogenes, Clostridium chauvoei, Campylobacter spp., Chlamydia psittaci, Anaplasma marginale, Leptospira spp. Causes leptospirosis, Bordetella bronchiseptica, Clostridium perfringens, Staphylococcus intermedins, Bartonella henselae, Chlamydophila felis, Mycoplasma spp, Pasteurella multocida, Erwinia amylovora, Xanthomonas spp., Xanthomonas axonopodis, Ralstonia solanacearum, Peclobacterium and Dickeya spp., Agrobaclerium tumefaciens, Pseudomonas syringae, Pseudomonas syringae pv. lachrymans, Xanthomonas campestris pv. campeslris, Xanthomonas oryzae, Clavibacler michiganensis, Xylella fastidiosa, Pantoea spp., Burkholderia glumae, Xanthomonas citri, Xanthomonas translucens, Xanthomonas fragariae, Xanthomonas hortorum, Clavibacter michiganensis, Pseudomonas syringae or a combination thereof.

[0114] In another example, the nucleic acid is a RNA or a DNA from a virus as disclosed herein, or a RNA or a DNA from a bacteria as disclosed herein; the RNA is reverse-transcribed to complementary DNA (cDNA), and then isothermally amplified using methods such as Recombinase Polymerase Amplification (RPA) or Loop-Mediated Isothermal Amplification (LAMP), before subjected to the method as disclosed herein. In another example, the nucleic acid is a cDNA. In another example, the target DNA is selected from the group consisting of single-stranded DNA, double- stranded DNA, or a combination thereof. In another example, the target DNA is a synthetic or naturally occurring DNA. In another example, the target DNA comprises a wild-type or mutant DNA. In another example, the target DNA includes DNA obtained by reverse transcription or amplification of RNA, such as cDNA.

[0115] Industrial Applicability

[0116] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.

[0117] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a primer” includes a plurality of primers, including mixtures and combinations thereof.

[0118] As used herein, the term “comprising” means “including.” Variations of the word "comprising", such as “comprise” and “comprises,” have correspondingly varied meanings. Thus, for example, a composition “comprising” X may consist exclusively of X or may include one or more additional unrecited components.

[0119] As used herein, the term “about” in the context of concentration of a substance, size of a substance, length of time, or other stated values means + / - 5% of the stated value, or + / - 4% of the stated value, or + / - 3% of the stated value, or + / - 2% of the stated value, or + / - 1% of the stated value, or + / - 0.5% of the stated value.

[0120] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0121] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0122] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also formpart of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0123] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

[0124] Other embodiments are within the following claims and non-limiting examples. EXAMPLES

[0125] Non-limiting examples of the disclosure will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the disclosure.

[0126] Example 1: Expression and Purification of Casl3d (Cdl4) enzyme

[0127] The Casl3 enzymes (Cdl3, Cdl4, and Cdl5) (disclosed inPCT / SG2022 / 050859, sequence can be seen in Table 1) were engineered with a dual affinity tag system: an N-tcrminal His6-MBP (hcxa-histidinc tag for purification and maltose binding protein for solubility enhancement) with a Tobacco Etch Virus (TEV) protease cleavage site, and a C-tcrminal enterokinase (EK) cleavage site followed by another His6 tag. These constructs were synthesized by GenScript (USA) and cloned into the pET21a(+) vector between the Ndel and Notl restriction sites. The resulting plasmids, pET21a(+)-His6-MBP-TEV-Casl3-EK-His6, was then introduced into Escherichia coli BL21 (DE3) cells for protein production.

[0128] Table 1. The list of sequences for Casl3 enzymes.

[0129] Protein expression was performed using 200 ml of Terrific broth in a lOOOcc flask. The E. coli cells harboring the plasmid were cultured at 37 °C with shaking at 180 rpm. When the optical density of the culture reached between 0.6 and 0.8, production of the Casl3 enzymes was induced with 400 pM IPTG. The culture was then shifted to a lower temperature (16-18°C) and allowed to grow overnight to express the protein.

[0130] After the overnight induction, cells were harvested and resuspended in a lysis buffer containing 50 mM Tris-HCl (pH 7.0), 500 mM NaCl, 5% glycerol, 1 mM Tris(2- carboxyethyl)phosphine (TCEP), and 0.5 mM Phenylmethyl sulfonyl fluoride (PMSF), supplemented with an EDTA-frcc protease inhibitor cocktail from Roche. To aid in cell disruption, lysozyme was added to the lysate at a concentration of 0.1-1 mg / ml, along with 500 mg to 1 g / L of protamine sulfate. This mixture was incubated on ice for 30 minutes with gentle swirling for homogeneous mixing.

[0131] The cells were subsequently lysed by sonication, performed on ice to prevent overheating, with settings adjusted to 60% amplitude, 0.5 cycle, and a pulse rate of 360 seconds on. Cell debris was then eliminated by centrifugation at 14,000 g for 30 minutes at 4°C, followed by filtration through a 0.45-micron filter.

[0132] The clarified cell lysate was processed through an affinity chromatography column loaded with Ni-NTA resin from Sigma- Aldrich at a proportion of lec resin per lOcc of cell lysate. The column was washed with a buffer containing 50 mM sodiumphosphate (pH 8.0), 300 mM NaCl, and 10 mM Imidazole. The Casl3 protein was then eluted using a buffer with elevated imidazole concentration (300 mM) and TCEP. The lysate, flow-through, wash, and elution fractions were all separately collected and assessed by SDS-PAGE for protein purity.

[0133] For protein concentration, fractions containing Casl3 were processed using Amicon filters with a 3k or 30k molecular weight cut-off. The concentrated enzyme was then incubated with TEV and EK proteases to remove the affinity tags. This step was performed at 4°C overnight while dialyzing against an ion-exchange buffer (50 mM Tris- HC1 pH 7.0, 250 mM KC1, 5% glycerol, 1 mM TCEP) to facilitate tag cleavage.

[0134] The tag-free Casl3 enzymes were then applied to an Amicon Ultra-0.5ml centrifugal Filter Ultracel-50k (Merck Millipore) for final purification. The enzyme solution was finally equilibrated with a storage buffer optimized for enzyme stability and activity, which included 20 mM Tris-HCl (pH 7.0), 200 mM KC1, 5% glycerol, and 1 mM TCEP.

[0135] The final products, post-protease treatment and concentration, were verified for purity and integrity by SDS-PAGE analysis, confirming the successful expression and purification of the Casl3 enzyme suitable for downstream enzymatic assays.

[0136] Example 2: Reaction Buffer Preparation

[0137] Base Reaction Buffer was composed of 20 mM HEPES, 50 mM KC1, 5 mM MgCl2, and 5% glycerol, pH=6.8. All salts, organic solvents, and proteins used in the experiment were of analytical grade and obtained from commercial sources (Table 2). These reagents were added to the Base Reaction Buffer in various combinations as described in the subsequent Examples. Each component was measured and mixed thoroughly to ensure homogeneity of the buffer solutions.

[0138] Table 2- The list of chemicals used in this study

[0139] Example 3: Nucleic acid preparation

[0140] The ability of buffers of this invention to enhance the CRISPR-Casl3-Cdl4 based detection was tested. To address this, the in vitro collateral cleavage activity of Casl3 enzymes were evaluated using an artificial target.

[0141] Artificial target sequence with cither a C, G, A or U protospaccr flanking sequence (PFS) were designed. DNA oligo templates for T7 transcription were synthesised and cloned in pUC57 by GenScript (Table 3).

[0142] Table 3. DNA fragments for production of RNA substrates (i.e. target sequences)

[0143] Phusion™ High-Fidelity PCR Master Mix (2X) (Thermo Fisher Scientific) was used to amplify the template DNA with forward primer (TAATACGACTCACTATAG) (SEQ ID NO: 11) and reverse (CTTTATGCTTCCGGCTCG) primer (SEQ ID NO: 12). The PCR reaction was assembled in a 25-μL volume following the manufacturer’s instructions, using cycling conditions recommended by the supplier, with an annealing temperature of 55°C and 35 cycles. The integrity of the amplified sequence was confirmed by Sanger sequencing. The PCR product was analyzed on a 2% agarose gel, and the target band was extracted using the GeneJET™ Gel Extraction Kit (Thermo Fisher Scientific). To verify successful recovery, 3 μL of the purified DNA was run on a 2% agarose gel. The DNA concentration was measured using a Picodrop and Qubit Fluorometer 4.

[0144] For single- stranded RNA (ssRNA) synthesis, the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) was used, with the T7 RNA polymeraseforward primer facilitating transcription. The IVT reaction was set up at room temperature according to the manufacturer’s protocol, using 1 pg of template DNA, followed by thorough mixing and brief centrifugation. The reaction was incubated at 37°C for 4 hours in a water bath.

[0145] To prevent template DNA contamination in downstream applications, DNase I digestion was performed immediately after the IVT reaction. 1 μL of DNase I (1 U / μL, included in the kit) was added to the reaction mix and incubated at 37°C for 15 minutes.

[0146] For RNA analysis, 0.5 μL of the IVT product was diluted in 10 μL of DEPC- treated water. Then, 10 μL of this diluted RNA was mixed with 10 μL of 2X RNA Loading Dye Solution (provided in the kit), incubated at 70°C for 10 minutes, and then chilled on ice before loading onto a 2% agarose gel. The RNA samples were analyzed against an RNA ladder for size verification.

[0147] RNA purification from IVT reaction was performed using GeneJET RNA Cleanup and Concentration kit (Thermo Fisher Scientific) as instructed by manufacturer. The purified RNA was stored at -80°C until use. 0.5 μL of the purified IVT product was diluted in 10 μL of DEPC-treated water and 10 μL of the diluted sample was mixed with 10 μL of 2X RNA Loading Dye Solution and was heated the sample at 70°C for 10 minutes and was chilled on ice prior to loading on a 2% Agarose Gel against an RNA Ladder.

[0148] Example 4. Ionic Influence on Casl3-Cdl4 Activity under Varying Salt Conditions

[0149] The activity of Casl3-Cdl4, similar to other enzymes, is known to be influenced by various factors, including the ionic environment in which it operates. Ions can affect the folding, stability, and catalytic activity of RNA and proteins by altering the electrostatic interactions and the structural conformation of biomolecules. Therefore, understanding the impact of different ionic conditions on Casl3-Cdl4 activity is crucial for optimizing its performance in various applications.

[0150] In this study, the aim was to investigate how the addition of various salts to the reaction buffer alters the activity of Casl3-Cdl4. The rationale behind this examination is rooted in the premise that ions play a significant role in enzymatic activity and may either enhance or inhibit the catalytic function of Casl3d. By systematically varying thetype and concentration of salts in the reaction buffer, it was aimed to delineate the optimal conditions that support the enzymatic activity of Casl3-Cdl4.

[0151] Methods

[0152] The base reaction buffer was composed of 20 mM HEPES, 50 mM KC1, 5 mM MgCl2, and 5% glycerol, pH=6.8. This buffer served as the control environment for all subsequent experiments.

[0153] A range of 10, 20, 40, and 60 mM of salts including KH2PO4, CdSO4, CO(NO3)2, HCOONa, Ca(C2H3O2)2, CHUOONa, (NH4)2SO4, AlCh, KSCN, FeSO4, CuSO4, LiSO4, A12(SO4)3, Ni(NO3)2, ZnCl2, CaCl2, NaCl, and K2SO4were added to the base reaction buffer. The final concentrations of the salts were varied in a systematic manner to assess the influence of ionic strength and salt composition on Casl3-Cdl4 activity.

[0154] For collateral cleavage assay, a 20μL detection system was prepared that consisted of 100 nM Casl3-Cdl4, 100 nM crRNA (CAACUACAACCCUGUCAAAUUACAGGGUUCUGAAACUAGAUUGCUGUUC UACCAAGUAAUCCAU) (SEQ ID NO: 13), 250nM ssRNA reporter ( / 56- FAM / rUrUrUrUrU / 3IABkFQ / ), and 250nM target ssRNA ((an equal mixture of the four targets listed in Table 3) in a lx cleavage buffer. Fluorescence readouts were taken in 1 min intervals for a total of 60 min on the ABI Real-time PCR (Applied Biosystems, CA, USA). The fluorescence signal, indicative of Casl 3-Cd14 activity, was recorded at regular intervals for a period of 1 hour. Since the enzyme activity for each test reached to its peak before 5 minutes, the activity of enzyme at 5 minute is shown in the graphs as means ± SD (n = 3). To facilitate comparisons across varying experimental conditions, fluorescence for background conditions (no target ssRNA) were subtracted from samples to generate background subtracted fluorescence.

[0155] Results

[0156] The addition of different salts to the reaction buffer resulted in varied effects on Casl3-Cdl4 activity, as shown in Figure 1. KH2PO4, HCOONa, Ca(C2H3O2)2, CH3COONa, (NH4)2SO4, KSCN, CaCl2, and NaCl enhanced the activity of Casl3-Cdl4, while CdSO4, Co(NO3)2, AICI3, CuSO4, EiSO4, A12(SO4)3, Ni(NO3)2, ZnCl2. FeSO4, and K2SO4inhibited the activity of enzyme. The data indicate a clear correlation between salt composition and Casl3-Cdl4 activity.

[0157] The addition of potassium thiocyanate (KSCN), sodium formate (HCOONa), monopotassium phosphate (KH2PO4), sodium acetate (CH3COONa), calcium acetate (C (C2H3O2)2), ammonium sulfate ((NH4)2SO4) resulted in an impressive 2.70-, 2.67-, 2.24-, 2.21-, 2.03-, and 1.88-fold increase in Casl3-Cdl4 activity, respectively.

[0158] In summary, the present results demonstrated that Casl3 activity was sensitive to the ionic composition of the reaction buffer. Both enhancing and inhibiting effects were observed depending on the type and concentration of the salt added. These findings could inform the optimization of reaction conditions for Casl3-based applications.

[0159] Example 5: The Influence of Organic Compounds on Casl3-Cdl4 Activity

[0160] This experiment was designed to assess the influence of different organic compounds at variable concentrations on the enzymatic activity of Casl3-Cdl4, providing insight into the optimal conditions for its function and potential regulatory mechanisms.

[0161] Methods

[0162] The base reaction buffer was as described in Example 3. A set of organic compounds was prepared at varying concentrations. Each compound was added to the reaction mix to achieve the final testing concentrations. A control without any added compounds was also maintained.

[0163] Fluorescence signals were monitored at regular intervals over an hour to measure Casl3d activity. As peak enzyme activity was achieved within 5 minutes in each assay, the graphs present the activity levels at the 5-minute mark as mean values with their standard deviations (means ± SD; n = 3). Baseline activity was established using the control reaction mix.

[0164] Results

[0165] It was observed that certain organic compounds enhanced Cas 13-Cdl4 activity at different concentrations (Figure 2). It was observed enhanced activity of Casl3-Cdl4 by introduction of Tween 20, Dithiothreitol (DTT), Phenylmethylsulfonyl fluoride (PMSF), 2-Methyl-2,4-pentanediol (MPD), and acetone. Some compounds such as EDTA and Urea did not exhibit a significant impact on enzymatic activity when compared with the control.

[0166] Example 6. Assessing the impact of Casein on Casl3-Cdl4 Activity

[0167] Adding a protein to an enzyme’s reaction buffer may enhance the enzyme's activity through various mechanisms. The added protein can act as a cofactor, facilitating the enzyme's ability to bind to its substrate or aiding in the catalytic process. It might also stabilize the enzyme's structure, maintaining it in an optimal conformation for activity or protecting it from denaturation and degradation. In some cases, the protein may interact with the enzyme to form a complex that has greater activity than the enzyme alone, or it might block the action of inhibitors, thereby sustaining the enzyme's function. Here the strategy of adding proteins to Casl3-Cdl4 reaction buffers to modulate enzyme activities in biochemical assays was examined. Given the lack of existing data on the relationship between casein and Casl3-Cdl4, the aim was to elucidate the potential modulatory effects of casein on the collateral cleavage activity of Casl3-Cdl4.

[0168] Methods

[0169] To investigate the influence of Casein on Casl3-Cdl4, casein with the concentration of 0.005, 0.01, 0.03, and 0.05 mg / tnl were added to control reaction buffer. The experiment and data analysis were performed as explained in Example 4.

[0170] Results

[0171] Incorporation of casein into the Casl3-Cdl4 reaction system revealed a notable concentration-dependent enhancement of enzymatic activity (Figure 3). Without casein, Casl 3-Cdl4 displayed a baseline activity with an average background subtracted fluorescence intensity of 108K. Upon introducing 0.005 mg / ml of casein, a 1.9-fold increase in enzymatic activity was observed. Further increase in casein concentration to 0.01 and 0.03 mg / ml resulted in 2.8- and 3-fold increases in fluorescence intensities, respectively, over the control. The signal intensity slightly declined and showed 1.2-fold increase compared the control when the concentration of casein increased to 0.05 mg / ml. These findings suggested a stimulatory role for casein in the context of Casl3-Cdl4 activity.

[0172] Example 7. Ionic Influence on Casl3-Cdl4 Activity in SARS-CoV-2 Detection

[0173] The ability of buffers of this invention to enhance the SARS-CoV-2 detection using CRISPR-Casl3-Cdl4 was tested.

[0174] Methods

[0175] A segment of the SARS-CoV-2 N gene (Table 4) (Broughton JP et al.'s study on CRlSPR-Casl2-based detection of SARS-CoV-2 (Nat Biotechnol. 2020 Jul;38(7):870-874), designated as the target, was artificially synthesized and then inserted into the pUC57 vector by GcnScript. This cloned fragment served as the template for subsequent T7 in-vitro transcription (IVT) to generate single- stranded RNA (ssRNA) templates. Single-stranded RNA (ssRNA) preparation and analysis were performed as described in Example 3.

[0176] Table 4. Sense DNA template of SARS-CoV-2 N gene for production of RNA substrates (i.e. target sequences)

[0177] The base reaction buffer consisted of 20 mM HEPES, 50 mM KC1, 5 mM MgCl2, and 5% glycerol (pH 6.8), serving as the control environment for all subsequent experiments. To evaluate the impact of ionic strength and salt composition on Casl3- Cdl4 activity, varying concentrations (0.25, 0.5, 1, 2.5, 5 and 10 mM) of Li2SO4, A12(SO4)3, (Ni(NO3)2,) ZnCl2, CaCl2, and NaCI were systematically added to the base reaction buffer. Since most of the salts tested in the previous experiment (Example 4) exhibited the highest enhancement of activity at lower concentrations (10 mM), a range of salt concentrations from 0.25 mM to 10 mM was examined in this experiment.

[0178] For the collateral cleavage assay, a 20 μL detection system was prepared, containing 100 nM Casl3-Cdl4, 50 nM crRNA (Table 5), 250 nM ssRNA reporter ( / 56- FAM / rUrUrUrUrU / 3IABkFQ / ), and 250 nM target ssRNA (an equal mixture of the fourtargets listed in Table 3) in a lx cleavage buffer. Fluorescence signals were recorded at 1-minute intervals for a total of 60 minutes using an ABI Real-Time PCR system (Applied Biosystems, CA, USA). Since enzyme activity peaked within the first 5 minutes, fluorescence measurements at the 5-minute mark are presented in the graphs as means ± SD (n=3). To ensure consistency across experimental conditions, background fluorescence (from reactions without target ssRNA) was subtracted from all samples to generate background-corrected fluorescence values.

[0179] Table 5. crRNA sequences (5’ to 3’) for SARS-CoV-2 detection.

[0180] Results

[0181] The addition of different salts to the reaction buffer resulted in varied effects on Casl3-Cdl4 activity. (Ni(NO3)2.) LiSO4. CaCl2, and NaCl enhanced the activity of Casl3-Cdl4, while Al2(SO4)3and ZnCl2, inhibited the activity of enzyme. The data indicate a clear correlation between salt composition and Casl3-Cdl4 activity (Figure 4).

[0182] The addition of Ni(NO3)2, LiSO4, CaCl2, and NaCl resulted in an impressive 3.9-, 2.6-, 4.0-, and 2.9-fold increase in Casl3-Cdl4 activity, respectively.

[0183] In summary, the present results demonstrated that Casl3-Cdl4-based disease detection can be optimized by modulating the ionic composition of the reaction buffer. The effects varied depending on the type and concentration of the salt, with both enhancement and inhibition observed. Notably, Ni(NO3)2enhanced Casl3-Cdl4 activity at lower concentrations, suggesting that nickel ions may play a role in stabilizing the enzyme’s active conformation, facilitating RNA binding, or improving the interaction between the enzyme and its crRNA-target complex. However, at higher concentrations, potential inhibitory effects may arise due to enzyme structural perturbation or nonspecific ionic interactions interfering with the cleavage mechanism.

[0184] Example 8. The Influence of Organic Compounds on Casl3-Cdl4 activity in SARS-CoV-2 detection

[0185] This experiment aimed to evaluate the impact of various organic compounds at different concentrations on the Casl3-Cdl4 base SARS-CoV-2 detection.

[0186] Methods

[0187] The base reaction buffer was prepared as described in Example 7. A range of organic compounds (DMSO, Tween 20, MPD, PMSF, propanol, acetone, EDTA, Urea, ethylene glycol, and methanol) was added to the reaction mix at varying concentrations, with a control reaction maintained without any additional compounds.

[0188] Collateral cleavage assay was performed as Example 7. Fluorescence signals were monitored at regular intervals over an hour to assess Casl3-Cdl4 activity. Since peak activity was consistently observed within the first 5 minutes, the data presented in the graphs reflect the enzyme activity at the 5-minute mark as mean values with standard deviations (mean ± SD; n = 3). Baseline activity was established using the control reaction mix.

[0189] Results

[0190] Organic compounds generally enhanced Casl3-Cdl4 activity for SARS-CoV- 2 detection (Figure 5). Notable enhancements were observed with DMSO, Tween 20, and ethylene glycol, which increased Casl3-Cdl4 activity by up to 6.7, 5.8, and 5.5 times, respectively. Additionally, MPD, PMSF, propanol, acetone, EDTA, urea, and methanol also significantly enhanced enzyme activity by up to 3.6, 2.6, 3.5, 3.2, 3.5, 2.2, and 3.9 times, respectively.

[0191] The effect of these organic compounds was concentration-dependent: while PMSF and DMSO increased Casl3-Cdl4 activity at higher concentrations, compounds such as Tween 20, propanol, methanol, and EDTA enhanced the activity at lower concentrations. In contrast, ethylene glycol significantly altered Casl3-Cdl4 activity at mid-range concentrations.

[0192] Example 9. The impact of Casein on Casl3-Cdl4 activity in SARS-CoV-2 detection

[0193] Here the strategy of adding casein to Casl3-Cdl4 reaction buffers to enhance the activity of enzyme for SARS-CoV-2 detection was examined.

[0194] Methods

[0195] To investigate the influence of Casein on Casl3-Cdl4 based SARS-CoV-2 detection, casein with the concentration of 0.0015, 0.003, 0.006, 0.015, and 0.03, and 0.06 mg / ml were added to control reaction buffer. The experiment and data analysis were performed as explained in Example 4.

[0196] Results

[0197] Incorporation of casein into the Cas 13 -Cd 14 reaction system revealed a notable concentration-dependent enhancement of enzymatic activity (Figure 6). Without casein, Cas 13 -Cd 14 displayed a baseline activity with an average background subtracted fluorescence intensity of around 38K. Upon introducing 0.015 mg / ml of casein, 6.3-fold increase in enzymatic activity was observed. Further increases in casein concentration did not result in additional fluorescence intensity beyond 0.015 mg / mL of casein. These findings suggest a stimulatory role for casein in the context of Casl3-Cdl4 activity.

[0198] Example 10. Limit of Detection (LOD) Assay

[0199] The limit of detection (LOD) is a critical evaluation of CRISPR-Casl3 sensitivity in identifying target nucleic acids. This assessment highlights Casl3-Cdl4 enzyme’s potential for sensitive SARS-CoV-2 detection diagnostics. To enhance the sensitivity of enzyme detection, casein was added to buffer.

[0200] Methods

[0201] In this experiment, the N-gene of SARS-CoV-2 was selected as the target sequence. Primers were designed using Oligo7 software, incorporating a T7 promoter sequence in the forward primer to enable in vitro transcription (IVT).

[0202] The target plasmid (pUC19+N-Gcnc) was synthesized by GcnScript. Plasmid concentration was measured using an Invitrogen Qubit 4 Fluorometer, following the manufacturer’s instructions. The DNA copy number per microliter was calculated using the formula:

[0203] DNA copy number per μL = [(6.02 x 1023) x (plasmid concentration in ng / μL) x 10,J] / [(fragment length in nucleotides) x 660]

[0204] The plasmid was serially diluted from 0 to 1 x 103copies / μL, with the following concentrations: negative control (0), 1, 5, 10, 100, and 1x 103copies / μL. The sensitivity of the RPA reaction was assessed using each dilution as a template, while RNase-free water served as a negative control.

[0205] The RPA reaction was conducted in a 50 μL final volume, with plasmid DNA concentrations ranging from 20 copies / μL to 0.02 copies / μL, based on the dilution of the plasmid stock and the addition of 1 μL of plasmid to the reaction. The reaction mixture contained 2.4 μL of each forward and reverse RPA primer (10 pM) (Table 6), 29.5 μLprimer-free rehydration buffer, 2.5 mM MgOAc, 1 uL target DNA, and RNase-free water to bring the total volume to 50 μL. The reaction was incubated in a 39°C water bath for 60 minutes.

[0206] Table 6. RPA Primer sequences (5' to 3')

[0207] Following RPA, IVT was performed to generate RNA from the amplified DNA. The IVT reaction had a total volume of 20 μL and contained 8 μL ATP / CTP / GTP / UTP mix, 4 μL 5X TranscriptAid Reaction Buffer, 2 μL TranscriptAid Enzyme Mix, and 6 μL of RPA product. The mixture was incubated in a 37°C water bath for 60 minutes.

[0208] The Casl3-Cdl4 collateral cleavage assay was performed in a 20 μL reaction volume, with three replicates for both positive and negative samples. The reaction mixture included 4 μL of 5X cleavage buffer, 0.015 mg / ml casein, 200 nM 264 Casl3 enzyme, 200 nM crRNA, 250 nM ssRNA fluorescence probe ( / 56-FAM / rUrUrUrUrU / 3IABkFQ / ), 0.25 μL IVT product (for the positive reaction), 1 μL DEPC-treated water (for the negative control), and RNase-free water to reach a total volume of 20 μL.

[0209] Real-time fluorescence measurements were collected on Step One ABI Realtime PCR (Applied Biosystems, CA, USA) at 1 min intervals for a total of 60 min. To allow comparisons between different conditions, fluorescence for background conditions (no target) were subtracted from samples to generate background subtracted fluorescence. The measurement of real-time background subtracted fluorescence output are shown in the graphs as means (n = 3).

[0210] Results

[0211] The sensitivity RPA amplification and IVT coupled with combination of Casl3-Cdl4 enzyme detection was tested here using a series of diluted positive quality controls (Figures 7).

[0212] The effective DNA copy number available in the Casl3-Cdl4 colateral cleavage reaction was determined by accounting for dilution factors across both the RPAand IVT steps. Starting with the initial DNA copies in the RPA reaction, 6 p L of the 50 u L RPA product was used for IVT. Following IVT, 1 μL of the 20 μL IVT product was transferred to the Casl3 reaction. The final effective DNA copies in the Casl3 assay were calculated using the formula:

[0213] Effective DNA copies = Initial DNA copies in RPA x 6 / 50 x 0.25 / 20

[0214] Through these sequential steps — RPA amplification, IVT conversion to RNA, and subsequent dilution into the Casl3 reaction — the effective DNA copies reaching Casl3 were calculated as follows: for initial DNA inputs of 1, 5, 10, 100, and 1000 copies in RPA, the final effective copies in Casl3 were 0.0015, 0.0075, 0.015, 015, and 1.5, respectively.

[0215] Casl3-Cdl4 demonstrated strong sensitivity, with fluorescence signals at 0.006 copies / μL significantly higher than the background (0 copies / μL). This highlights its ability to reliably detect very low concentration of DNA.

[0216] Example 11. The Influence of compounds on Casl3-Cdl3 and Casl3-Cdl5 activity in SARS-CoV-2 detection

[0217] This experiment aimed to assess the effect of three compounds at varying concentrations on two different Casl3 enzymes, Casl3-Cdl3 and Casl3-Cdl5, for SARS-CoV-2 detection. The goal was to determine whether the enhancement observed with these compounds in one enzyme could be replicated in others.

[0218] Methods

[0219] The base reaction buffer was prepared as described in Example 7. The best concentration of Tween 20 (0.25%V / V), MPD (2.5%V / V), and casein (0.015 mg / ml) observed in Examples 8 and 9 were added to the reaction mix, with a control reaction maintained without any additional compounds.

[0220] For the collateral cleavage assay, a 20 μL detection system was prepared, containing 200 nM Casl3-Cdl3 or Casl3-Cdl5, 200 nM crRNA (Table 7), 250 nM ssRNA reporter ( / 56-FAM / rArArArArA / 3IABkFQ / ), and 250 nM target ssRNA in a lx cleavage buffer. Fluorescence signals were recorded at 1 -minute intervals for a total of 60 minutes using an ABI Real-Time PCR system (Applied Biosystems, CA, USA). Since enzyme activity peaked within the first 5 minutes, fluorescence measurements at the 5- minute mark are presented in the graphs as means ± SD (n = 3).

[0221] Fluorescence signals were monitored at regular intervals over an hour to assess Casl3-Cdl4 activity. Since peak activity was consistently observed within the fust 5 minutes, the data presented in the graphs reflect the enzyme activity at the 5-minute mark as mean values with standard deviations (mean ± SD; n = 3). Baseline activity was established using the control reaction mix.

[0222] Table 7. crRNA sequences (5’ to 3’) for for SARS-CoV-2 detection.

[0223] Results

[0224] To determine whether the tested compounds could also enhance the activity of other Casl3 enzymes, the effects of Tween 20, MPD, and Casein on two additional Casl3 enzymes, Casl3-Cdl3 and Casl3-Cdl5 were examined. The present results showed that all three compounds significantly enhanced the activity of these enzymes (Figure 8). Notably, Tween 20 and Casein led to remarkable activity increases in Casl3-Cdl5, enhancing its fluorescence signal by 10.4-fold and 11.2-fold, respectively. These findings suggest that the enhancement effect of these compounds is not limited to a single Casl3 variant but may be broadly applicable across different members of the Casl3 family, potentially improving the sensitivity and efficiency of CRISPR-Casl3-based detection systems.

[0225] The present disclosure has focused on examining the influence of various compounds on the activity of a singular Casl3d enzyme. However, the scope of the present inquiry extends beyond this specific interaction. It is essential to consider that these compounds may also affect the activity of other enzymes in the Casl3 family, each of which could respond differently to the same molecular interventions. Furthermore, the potential impact of these compounds on Casl2 enzymes should not be overlooked, as Cas 12 enzymes possess collateral activities that are distinct from those of Cas 13 enzymes. These collateral activities are crucial for the CRISPR system's function in diagnostics. While the present experimental data currently demonstrate enhancement of Cas 13 enzymatic activity through the addition of novel buffer components, it can be reasonably anticipated that similar improvements may extend to Casl2 enzymes. This expectation is based on several shared mechanistic and structural features between Casl 3 and Cas12systems. Both enzymes are RNA-guided nucleases that require precise buffer conditions to maintain optimal tertiary conformation and catalytic activity. Furthermore, the identified compounds act through mechanisms such as stabilizing protein-RNA interactions, enhancing target binding, or facilitating ion-dependent conformational changes — features that arc also critical for Casl2 function. Given these mechanistic similarities, and the lack of reported data refuting such effects on Casl2, it is plausible that these novel buffer additives will likewise enhance Casl2 activity. This potential cross-applicability supports the broader utility of the present buffer system across the CRISPR-Cas enzyme family.References:• Dang S, Sui H, Zhang S, Wu D, Chen Z, Zhai J, Bai M. CRISPR-Casl2a test strip (CRISPR / CAST) package: In-situ detection of Brucella from infected livestock. BMC Veterinary Research. 2023 Oct 13 ; 19(1 ):202.• Xu J, Ma J , Li Y, Kang L, Yuan B, Li S, Chao J, Wang L, Wang J, Su S, Yuan Y. A general RPA-CRISPR / Casl2a sensing platform for Brucella spp. detection in blood and milk samples. Sensors and Actuators B: Chemical. 2022 Aug1 ;364: 131864.

Claims

Claims1. A buffer for enhancing enzymatic cleavage activity of a Cas protein, comprising one or more components selected from the group consisting of: a salt selected from the group consisting of monopotassium phosphate (KH2PO4), sodium formate (HCOONa), calcium acetate (Ca(C2H3O2)2), sodium acetate (CH3COONa), ammonium sulfate ((NH4SO4), potassium thiocyanate (KSCN), calcium chloride (CaCl2), sodium chloride (NaCl), Nickel(II) nitrate (NiCNChh), lithium sulfate (LiSO4); an organic solvent selected from the group consisting of Tween 20, Dithiothreitol (DTT), Phenylmethylsulfonyl fluoride (PMSF), 2-Methyl- 2,4-pentanediol (MPD), acetone, dimethylsulfoxide (DMSO), ethylene glycol, methanol, 2-propanol, EDTA, urea; and a protein which is casein.

2. The buffer of claim 1, further comprising a base composition comprising HEPES, KC1, MgCl2, and glycerol.

3. The buffer of claim 2, wherein the base composition is one of the following: a base composition comprising about 20 mM HEPES, about 50 mM KC1, about5 mM MgCl2, about 5% glycerol, and having a pH of about 6.8; a base composition comprising about 20 mM HEPES, about 50 mM NaCl, about 10 mM MgCl2. about 5% glycerol, and having a pH of about 7.0; a base composition comprising about 20 mM HEPES, about 50 mM NaCl, about 10 mM MgCl2, about 5% glycerol, about Ipg / ml BSA, and having a pH of about 7.0; a base composition comprising about 20 mM HEPES, about 60 mM NaCl, about6 mM MgCl2, about 5% glycerol, and having a pH of about 6.8; a base composition comprising about 20 mM HEPES, about 60 mM NaCl, about 5% PEG, about 5 pM DTT, and having a pH of about 6.8; anda base composition comprising about 20 mM HEPES, about 60 mM KC1, about 5% PEG, and having a pH of about pH 8.0.

4. The buffer of claim 1, wherein the Cas protein is selected from the group consisting of Casl3d, Casl3a, Casl3b, Casl3c, Casl2a, Casl2b, Casl2c, Casl2d, and Casl2f, optionally the Cas 13d protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, and 6.

5. The buffer of claim 1, wherein the concentration of each of the salt is 5 mM - 3 M, 10-60 mM, or 0.25-10 mM.

6. The buffer of claim 1, wherein the concentration of each of the organic solvent is 5 mM - 3 M, or 1-5 mM.

7. The buffer of claim 1, wherein the concentration of monopotassium phosphate (KH2PO4) is 10-60 mM.

8. The buffer of claim 1, wherein the concentration of sodium formate (HCOONa) is 10-60 mM.

9. The buffer of claim 1 , wherein the concentration of calcium acetate (Ca(C2H3O2)2) is 10-60 mM.

10. The buffer of claim 1 , wherein the concentration of sodium acetate (CH3COONa) is 10-60 mM.1 1 . The buffer of claim 1 , wherein the concentration of ammonium sulfate ((NH4)2SO4) is 10-60 mM.

12. The buffer of claim 1, wherein the concentration of potassium thiocyanate (KSCN) is 10-60 mM.

13. The buffer of claim 1, wherein the concentration of calcium chloride (CaCl2) is 10-60 mM, or 1.25-50 mM.

14. The buffer of claim 1, wherein the concentration of sodium chloride (NaCl) is0.25-10 mM, or 1.25-50 mM.

15. The buffer of claim 1, wherein the concentration of Nickel(II) nitrate (Ni(NO3)2is 0.25-10 mM.

16. The buffer of claim 1, wherein the concentration of lithium sulfate (LiSO4) is 0.25- 10 mM.

17. The buffer of claim 1, wherein the concentration of DTT is 0.1-15 mM, or 1-5 mM.

18. The buffer of claim 1, wherein the concentration of PMSF is 0.1-15 mM, 0.25-10 mM, or 1 -2.5 mM.

19. The buffer of claim 1, wherein the concentration of DMSO is 0.1-15 mM, 0.25- 10 mM, or 1 -2.5 mM.

20. The buffer of claim 1, wherein the concentration of Tween 20 is 0.1-15 mM, 0.25- 10 mM, or 2.5-5 mM.

21. The buffer of claim 1, wherein the concentration of ethylene glycol is 0.1-15 mM, 0.25-10 mM, or about 2.5 mM.

22. The buffer of claim 1, wherein the concentration of methanol is 0.1-10 mM, 0.25- 5 mM, or 1-5 mM.

23. The buffer of claim 1, wherein the concentration of 2-propanol is 0.1-15 mM, 0.25-10 mM, or 1-2.5 mM.

24. The buffer of claim 1, wherein the concentration of acetone is 0.1-15 mM, 0.25- 10 mM, or 1-2.5 mM.

25. The buffer of claim 1, wherein the concentration of MDP is 0.1-15 mM, 0.25-10 mM, or about 4 mM.

26. The buffer of claim 1, wherein the concentration of EDTA is 0.1-5 mM, or 0.25-1 mM.

27. The buffer of claim 1, wherein the concentration of urea is 0.1-15 mM, 0.25-10 mM.

28. The buffer of claim 1, wherein the concentration of casein is 0.001-10 mg / mL, or 0.01-1 mg / mL.

29. The buffer of claim 28, wherein the concentration of casein is 0.005-0.05 mg / mL, or 0.0015-0.06 mg / mL.

30. The buffer of any one of claims 1-29, wherein the buffer has a PH of between 6.0 and 8.0.

31. The buffer of any one of claims 1-30, wherein the enzymatic cleavage activity is increased by 1 .5-20 fold.

32. The buffer of claim 31, wherein the enzymatic cleavage activity is increased by 1.76-3.04 fold, or 1.88-2.70 fold, or 10.4-11.2 fold.

33. The buffer of claim 2, wherein the buffer comprises the base composition and one or more salts of claim 1.

34. The buffer to claim 2, wherein the buffer comprises the base composition and one or more organic solvents of claim 1.

35. The buffer to claim 2, wherein the buffer comprises the base composition and casein.

36. The buffer to claim 2, wherein the buffer comprises the base composition, one or more salts and one or more organic solvents of claim 1.

37. The buffer to claim 2, wherein the buffer comprises the base composition, one or more salts of claim 1, and casein.

38. The buffer to claim 2, wherein the buffer comprises the base composition, one or more organic solvents of claim 1, and casein.

39. The buffer to claim 2, wherein the buffer comprises the base composition, one or more salts of claim 1, one or more organic solvents of claim 1, and casein.

40. A detection kit for a nucleic acid, comprising:(a) the buffer of any one of claims 1-39;(b) a Cas protein selected from the group consisting of Casl3d, Casl3a, Casl3b, Casl3c, Casl2a, Casl2b, Casl2c, Casl2d, and Casl2f; and(c) a crRNA configured to target the nucleic acid and form a complex with the Cas protein.

41. The detection kit of claim 40, further comprising a detecting agent comprising a probe nucleic acid, wherein the detecting agent generates a detectable signal when the probe nucleic acid is cleaved by the Cas protein.

42. The detection kit of claim 41, wherein the probe nucleic acid is a fluorescent probe, wherein the 5' end of the fluorescent probe sequence is labeled with a fluorescent group, and the 3' end is labeled with a quenching group; or a colorimetric probe.

43. The detection kit of claim 42, wherein the fluorescent group is selected from the group consisting of FAM, VIC, HEX, TRT, Cy3, Cy5, ROX, JOE and Texas Red, and the quenching group is selected from the group consisting of DABCYL, MGB, BHQ-1, BHQ-2 and BHQ-3.

44. The detection kit of claim 42, wherein the colorimetric probe comprises FAM and Biotin, and is for detection by a lateral flow strip.

45. The detection kit of any one of claims 40-44, wherein the Cas protein and the crRNA arc in the form of a ribonuclcoprotcin complex.

46. The detection kit of any one of claims 40-45, wherein the Cas protein and the crRNA are lyophilized.

47. The detection kit of any one of claims 40-46, wherein shelf-life of the Cas protein and the crRNA is increased by 30-300% in the presence of the buffer of any oneof claims 1-39, compared to the self-life of the Cas protein and the crRNA in the absence of the buffer of any one of claims 1-39.

48. A method of detecting a nucleic acid in a sample by clustered regularly interspaced short palindromic repeats and CRISPR associated protein (CRISPR- cas), using the detection kit of any one of claims 40-47, the method comprising: incubating the sample containing the nucleic acid with the Cas protein and the crRNA, wherein when the nucleic acid is present in the sample, enzymatic cleavage activity of the Cas protein is induced to generate a detectable signal when the probe nucleic acid is cleaved; wherein the buffer enhances the enzymatic cleavage activity of the Cas protein.

49. The method of claim 48, further comprising amplifying the nucleic acid in the sample, using a technology selected from the group consisting of Polymerase Chain Reaction (PCR), Recombinase Polymerase Amplification (RPA), Loop- Mediated Isothermal Amplification (LAMP), and Rolling Circle Amplification (RCA).

50. The method of claim 48, wherein the method does not comprise amplifying the nucleic acid in the sample.

51. The method of claim 48, further comprising using a detection method selected from the group consisting of a colorimetric detection method, a fluorescent detection method and an electrochemical detection method to detect the detectable signal which is a colorimetric signal, a fluorescent signal, or an electrochemical signal.

52. The method of claim 51, wherein the colorimetric detection method uses a lateral flow strip and a colorimetric probe comprising FAM and Biotin.

53. The method of claim 51, wherein the fluorescent detection method uses a fluorescent probe, wherein the fluorescence signal is measured using a fluorometer, a microplate reader, or Real-Time PCR.

54. The method of claim 51 , wherein the electrochemical detection method transduces CRISPR-Cas cleavage event to an electrochemical signal using a modified electrode or a nanoparticle-based sensor.

55. The method of claim 48, wherein the sample is a biological, chemical or environmental sample.

56. The method of claim 55, wherein the biological sample is obtained from a tissue sample, saliva, blood, plasma, sera, stool, urine, sputum, mucous, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, or swab of skin or a mucosal membrane surface or a combination thereof.

57. The method of claim 55, wherein the chemical sample is obtained from a pharmaceutical substance, a chemical reagent, a cosmetic product, an industrial effluent, a pollutant, a hazardous material, a polymer, an adhesive, a solvent, an oil, a gas, a metal alloy, a nanomaterial, a fuel, a cleaning agent, a dye, or a combination thereof.

58. The method of claim 55, wherein the environmental sample is obtained from a food, a beverage, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a waste water sample, a saline water sample, exposure to atmospheric air or other gas sample, or a combination thereof.

59. The method of claim 55, wherein the biological, chemical or environmental sample is a crude sample and / or wherein the nucleic acid is not purified or amplified from the sample.

60. The method of any one of claims 48-59, wherein the sample contains a microbe selected from a bacterium, a virus, an archaea, a protozoa, a fungi, an algae, a slime mode, a lichen, and a prion.

61. The method of claim 60, wherein the virus is selected from a group consisting of a double- stranded DNA virus, a single- stranded DNA virus, a double -strandedRNA virus, a positive sense RNA virus, a negative sense RNA virus, and a retrovirus.

62. The method of claim 61, wherein the virus is selected from a group consisting of a coronavirus, an Ebola virus, measles, SARS, Chikungunya virus, Marburg, MERS, Dengue, Lassa, influenza, rhabdovirus, HIV, a hepatitis virus (including hepatitis A, B, C, D, or E), an influenza virus (including an influenza A or influenza B), a human respiratory syncytial virus, Sudan ebola virus, Bundibugyo virus, Tai Forest ebola virus, Reston ebola virus, Achimota virus, Aedes flavivirus, Aguacate virus, Akabane virus, Alethinophid reptarenavirus, Allpahuayo mammarenavirus, Amapari mmarenavirus, Andes virus, Apoi virus, Aravan vims, Aroa virus, Arumwot virus, Atlantic salmon paramyxovirus, Australian bat lyssavirus, Avian bomavirus, Avian metapneumovirus, Avian paramyxoviruses, penguin or Falkland Islandsvirus, BK polyomavirus, Bagaza virus, Banna virus, Bat herpesvirus, Bat sapovirus, Bear Canon mammarenavirus, Beilong virus, Betacoronavirus, Betapapillomavirus 1 -6, Bhanja virus, Bokeloh bat lyssavirus, Boma disease virus, Bourbon virus, Bovine hepacivirus, Bovine parainfluenza virus 3, Bovine respiratory syncytial virus, Brazoran vims, Bunyamwera virus, Caliciviridae virus. California encephalitis vims, Candiru vims, Canine distemper vims, Canine pneumovirus, Cedar virus, Cell fusing agent vims, Cetacean morbillivims, Chandipura vims, Chaoyang vims, Chapare mammarenavirus, Colobus monkey papillomavirus, Colorado tick fever virus, Cowpox virus, Crimean-Congo hemorrhagic fever vims, Culex flavivirus, Cupixi mammarenavirus, Dengue virus, Dobrava-Belgrade vims. Donggang vims, Dugbe virus, Duvenhage virus, Eastern equine encephalitis vims, Entebbe bat vims, Enterovirus A-D, European bat lyssavirus 1-2, Eyach virus, Feline morbillivims, Fer-de- Lance paramyxovirus, Fitzroy River vims, Flaviviridae vims, Flexal mammarenavirus, GB virus C, Gairo virus, Gemycircularvims, Goose paramyxovirus SF02, Great Island virus, Guanarito mammarenavirus, Hantaan virus. Hantavirus Z10, Heartland vims, Hendra vims, Hepatitis A / B / C / E, Hepatitis delta vims, Human bocavirus, Human coronavirus, Human endogenous retrovirus K, Human enteric coronavims, Human genital-associated circular DNA vims- 1, Human herpesvirus 1-8, Human mastadenovirus A-G, Humanpapillomavirus, Human parainfluenza virus 1-4, Human paraechovirus, Human picomavims, Human smacovirus, Ikoma lyssavirus, Ilheus virus. Influenza A-C, Ippy mammarenavirus, Irkut virus, J-virus, JC polyomavirus, Japanese encephalitis virus, Junin mammarenavirus, KI polyomavirus, Kadipiro virus, Kamiti River virus, Kcdougou virus, Khujand virus, Kokobcra virus, Kyasanur forest disease virus, Lagos bat virus, Langat virus, Lassa mammarenavirus, Latino mammarenavirus, Leopards Hill virus, Liao ning virus, Ljungan virus, Lloviu virus, Louping ill virus, Lujo mammarenavirus, Luna mammarenavirus, Lunk virus, Lymphocytic choriomeningitis mammarenavirus, Lyssavirus Ozemoe, MSSI2Y225 virus, Machupo mammarenavirus, Mamastrovirus 1, Manzanilla vims, Mapuera virus, Marburg virus, Mayaro vims, Measles virus, Menangle virus, Mercadeo virus, Merkel cell polyomavirus, Middle East respiratory syndrome coronavirus, Mobala mammarenavirus, Modoc virus, Moijang vims, Mokolo virus, Monkeypox virus, Montana myotis leukoenchalitis virus, Mopeia lassa virus reassortant 29, Mopeia mammarenavirus, Morogoro virus, Mossman vims, Mumps vims, Murine pneumonia virus, Murray Valley encephalitis vims, Nariva vims, Newcastle disease virus, Nipah vims, Norwalk vims, Norway rat hepacivims, Ntaya virus, O'nyong-nyong virus, Oliveros mammarenavirus, Omsk hemorrhagic fever virus, Oropouchc vims, Parainfluenza vims 5, Parana mammarenavirus, Parramatta River vims, Peste-des-petits- ruminants virus, Pichande mammarenavirus, Picomaviridae virus, Pirital mammarenavirus, Piscihepe virus A, Porcine parainfluenza vims 1, porcine rubulavims, Powassan vims, Primate T-lymphotropic vims 1-2, Primate erythroparvovims 1, Punta Toro vims, Puumala vims, Quang Binh virus, Rabies virus, Razdan virus, Reptile bomavims 1, Rhinovirus A-B, Rift Valley fever virus, Rinderpest vims, Rio Bravo virus, Rodent Torque Teno virus, Rodent hepacivirus, Ross River virus, Rotavirus A-I, Royal Farm virus, Rubella virus, Sabia mammarenavirus, Salem vims, Sandfly fever Naples virus, Sandfly fever Sicilian virus, Sapporo virus, Sathuperi vims, Seal anellovims, Semliki Forest virus, Sendai virus, Seoul virus, Sepik vims, Severe acute respiratory syndrome-related coronavirus. Severe fever with thrombocytopenia syndrome virus, Shamonda vims, Shimoni bat virus, Shuni vims, Simbu vims, Simian torque teno virus, Simian virus 40-41, Sin Nombrevirus, Sindbis virus, Small anellovirus, Sosuga virus, Spanish goat encephalitis virus, Spondweni virus, St. Louis encephalitis virus, Sunshine virus, TTV-like mini virus, Tacaribe mammarenavirus, Taila virus, Tamana bat virus, Tamiami mammarenavirus, Tembusu virus, Thogoto virus, Thottapalayam virus, Tick- bomc encephalitis virus, Tioman virus, Togaviridac virus, Torque teno canis virus, Torque teno douroucouli virus, Torque teno felis virus, Torque teno midi virus, Torque teno sus virus, Torque teno tamarin virus, Torque teno virus, Torque teno zalophus virus, Tuhoko virus, Tula virus, Tupaia paramyxovirus, Usutu virus, Uukuniemi virus, Vaccinia virus, Variola virus, Venezuelan Vesicular stomatitis Indiana virus, WU Polyomavirus, Wesselsbron virus, West Caucasian bat virus, West Nile virus, Western equine encephalitis vims, Whitewater Arroyo mammarenavirus, Yellow fever virus, Yokose virus, Yug Bogdanovac virus, Zaire ebolavirus, Zika virus, or Zygosaccharomyces bailii vims Z viral sequence, Canine Parvovirus, Canine Distemper Virus, Canine Coronavirus, Canine Influenza Virus, Feline Herpesvirus, Feline Calicivirus, Feline Leukemia Vims, Feline Immunodeficiency Virus, Canine Adenovirus - Type 1 and Type 2, Canine Herpesvirus, Tobacco Mosaic Virus, Potato Virus Y, Tomato Spotted Wilt Vims, Cucumber Mosaic Vims, Potato Virus X, Barley Yellow Dwarf Virus, Bean Common Mosaic Vims, Maize Dwarf Mosaic Vims, Soybean Mosaic Vims, Citrus Tristeza Virus, Rice Yellow Mottle Virus, Plum Pox Virus, Turnip Mosaic Vims, Aphid-Transmitted Yellow Dwarf Viruses, Cotton Leaf Curl Vims, Papaya Ringspot Virus, Cassava Mosaic Vims, Rice Tungro Virus, Apple Mosaic Vims, Sugarcane Mosaic Virus or a combination thereof.

63. The method of claim 62, wherein the coronavirus is SARS-CoV-2.

64. The method of claim 60, wherein the bacterium is a Gram-positive bacterium or a Gram-negative bacterium selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile and Clostridium tetani, Corynebacterium diphtheria, Listeria monocytogenes,Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Chlamydia trachomatis, Yersinia pestis, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, Salmonella typhi, and Vibrio cholera, Brucella abortus, Brucella melitensis, Brucella suis, Brucella canis, Brucella ovis, Brucella neotomae, Mycoplasma mycoides, Clostridium perfringens, Mycobacterium avium Salmonella spp., Pasteurella multocida, Haemophilus parasuis, Aclinobacillus pleuropneumoniae, Yersinia pestis, Prancisella tularensis, Bacillus anthracis, Coxiella burnetiid, Listeria monocytogenes, Clostridium chauvoei, Campylobacter spp., Chlamydia psittaci, Anaplasma marginale, Leptospira spp. Causes leptospirosis, Bordetella bronchiseptica, Clostridium perfringens, Staphylococcus intermed.ius, Bartonella henselae, Chlamydophila felis, Mycoplasma spp, Pasteurella multocida, Erwinia amylovora, Xanthomonas spp., Xanthomonas axonopodis, Ralstonia solanacearum, Pectobacterium and Dickeya spp., Agrobacterium tumefaciens, Pseudomonas syringae, Pseudomonas syringae pv. lachrymans, Xanthomonas campestris pv. campestris, Xanthomonas oryzae, Clavibacter michiganensis, Xylella fastidiosa, Pantoea spp., Burkholderia glumae, Xanthomonas citri, Xanthomonas translucens, Xanthomonas fragariae, Xanthomonas hortorum, Clavibacter michiganensis, Pseudomonas syringae or a combination thereof.

65. The method of any one of claims 48-64, wherein the method is performed at a temperature of 20-60 °C.

66. The method of claim 65, wherein the method is performed at a temperature of about 37 °C.

67. The method of claim 48, wherein a detection limit of the nucleic acid is at least 0.0015 DNA copies / μL.

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