Compositions and methods for instant nucleic acid detection

Abstract

Compositions and methods are provided for a simple, instrumentless and sensitive method that enables rapid, instant detection of nucleic acid molecules of interest. This is based on a surprising discovery that the relative efficiency of amplification and CRISPR-based cleavage and detection can be adjusted to facilitate amplification until sufficient amplification products are generated to achieve detection. Example methods include designing a guide RNA and a primer to target a non-optimal PAM sequence, or a sequence engineered Cas nuclease to reduce its activity in forming a ribonuclein with the guide RNA or binding or cleaving a substrate nucleic acid.

Description

This application is a divisional application of a patent application with an application date of January 7, 2022, application number 202280002216.4, and invention name “Compositions and methods for instant nucleic acid detection”. Technical Field The present invention relates to a composition and method for instant nucleic acid detection. Background Art

[0001] Over the past few decades, there have been several large-scale epidemic outbreaks caused by viruses (such as severe acute respiratory syndrome coronavirus (SARS), Middle East respiratory syndrome coronavirus (MERS), human immunodeficiency virus (HIV), Zika virus, Ebola virus), and the current pandemic outbreak caused by SARS-CoV-2. SARS-CoV-2 has spread rapidly around the world, in part due to the high prevalence of presymptomatic and asymptomatic transmission.

[0002] Nucleic acid diagnostic testing has limited capacity to slow transmission, as quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based testing, the gold standard for SARS-CoV-2 diagnosis, requires skilled personnel, equipment infrastructure, and long sample-to-answer times. A point-of-care nucleic acid test that is sensitive for detecting asymptomatic carriers and has a turnaround time fast enough to obtain results before clustering is essential. Isothermal amplification assays, such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), offer a rapid, instrument independent, and low-cost alternative compared to qPCR. However, nonspecific amplification of these assays can result in high false-positive rates.

[0003] The CRISPR / Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) is an RNA-guided endonuclease that has been used as a powerful genome editing tool. Due to the incidental degradation of ssDNA or ssRNA by Cas12a, Cas12b, and Cas13a, they have been repurposed as promising diagnostic tools. By amplifying the target sequence and performing sequential cleavage by Cas12 or Cas13, pathogens such as Zika virus and HPV can be detected at detection limits similar to qPCR.

[0004] These methods include the original two-step high-specificity, high-sensitivity enzymatic reporter (SHERLOCK) and DNA endonuclease-targeted CRISPR trans-reporter (DETECTR). Recently, both the two-step SHERLOCK and DETECTR have been clinically validated to detect SARS-CoV-2 with high sensitivity and reliability. However, the two-step method is complex and time-consuming to operate, and frequent opening of the lid increases the risk of contamination. STOP (SHERLOCK One-Pot Test) and SHINE (SHERLOCK and HUDSON Integrated for Epidemic Response) simply drop the simply extracted nucleic acid into a reaction mixture containing isothermal amplification and cleavage volumes, which are then read out by a fluorescent reader or strip. However, these assays typically require a total reaction time of about an hour. In contrast, Abbott's ID NOW COVID-19 test, which applies isothermal amplification to unextracted samples, is able to report results in less than 15 minutes, but it has considerable false-positive and false-negative rates. Summary of the invention

[0005] The inventors of the present invention have developed a one-step, rapid, sensitive, reliable and flexible nucleic acid detection assay. The assay shows a detection limit comparable to quantitative PCR (qPCR), but in a significantly shorter time, e.g., from 15 to 20 minutes. Thus, the present application provides a simple, instrument-free and sensitive alternative to the gold standard qPCR, and enables rapid, instant screening of target nucleic acid molecules.

[0006] One embodiment of the present disclosure provides a method for detecting a target polynucleotide, the method comprising incubating the target polynucleotide in a mixture comprising (a) a polymerase, (b) deoxynucleoside triphosphates (dNTPs), (c) primers for amplifying the target polynucleotide, (d) a CRISPR-associated (Cas) nuclease, and (d) a guide RNA comprising a spacer segment complementary to a target segment on the target polynucleotide, the incubation being performed under conditions such that the polymerase effectively amplifies the target polynucleotide, while the Cas nuclease is capable of cleaving the amplified target polynucleotide.

[0007] Also provided is a kit or package for detecting a target polynucleotide, the kit or package comprising (a) a polymerase, (b) deoxynucleoside triphosphates (dNTPs), (c) primers for amplifying the target polynucleotide, (d) a CRISPR-associated (Cas) nuclease, and (d) a guide RNA comprising a spacer segment complementary to a target segment on the target polynucleotide, wherein the polymerase can effectively amplify the target polynucleotide, and the Cas nuclease can cleave the amplified target polynucleotide.

[0008] Also provided is a kit or package for detecting a target polynucleotide, the kit or package comprising (a) a polymerase, (b) deoxynucleoside triphosphates (dNTPs), and (c) primers for amplifying the target polynucleotide, wherein at least one primer comprises a suboptimal PAM sequence for a Cas nuclease, or wherein DNA fragments amplified by the polymerase contain one or more suboptimal PAMs targeted by the Cas nuclease, or wherein at least the dNTPs or primers are modified to reduce cleavage or binding by the Cas nuclease.

[0009] In another embodiment, a kit or package for cleaving a target polynucleotide is further provided, the kit or package comprising (a) a CRISPR-associated (Cas) nuclease, and (b) a guide RNA comprising a spacer fragment complementary to a target fragment on the target polynucleotide, wherein the guide RNA has reduced binding to the target polynucleotide or reduced cleavage of the target polynucleotide compared to a standard guide RNA.

[0010] In another embodiment, a mutant Cas nuclease is also provided, which has (a) reduced activity in forming ribonucleoprotein (RNP), (b) an altered conformation, (c) reduced activity in interacting with a target PAM sequence, or (d) reduced binding to a target polynucleotide to be cleaved. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Suboptimal PAMs mediate faster one-pot reactions than canonical PAMs. (ad) Fluorescence signals of spacer 4 and spacer 5 of the Orf1ab gene in the incidental activity assay (ab) and one-pot reaction (cd) at 37°C. Suboptimal PAMs of spacer 4 (GTTG) and spacer 5 (CTTA) of Orf1ab were mutated to canonical PAMs of spacer 4 (TTTG) and spacer 5 (TTTA), respectively. (eh) Schematic diagram of the fluorescence kinetics of suboptimal PAMs and three canonical PAMs with point mutations at positions 1-3 in the incidental activity assay (e and f) and the corresponding one-pot reactions of spacer 4 (g) and spacer 5 (h). The time to reach half-maximal fluorescence was determined. Fluorescence values ​​for the incidental activity and one-pot reaction were determined at 40 min and 20 min, respectively. (n=3). The concentration of dsDNA substrate was 3.5 nM in the incidental activity assay and 2340 fM in the one-pot reaction.

[0012] Figure 2Sensitivity and reliability of one-pot reactions mediated by suboptimal PAMs. The sensitivity and reliability of one-pot reactions using suboptimal PAMs and classical PAMs were compared. crRNAs targeting the Orf1ab gene (spacers 4 and 5) and the envelope (E) gene (spacer 8) of SARS-CoV-2 were used. (ac) Sensitivity (ab) and reliability (c) of spacer 4 using suboptimal PAMs and classical PAMs. (df) Sensitivity (de) and reliability (f) of spacer 5 using suboptimal PAMs and classical PAMs. (gi) Sensitivity (gh) and reliability (i) of spacer 8 using suboptimal PAMs and classical PAMs. The substrate concentrations in c, f, and i were 2340fM, 2340fM, and 325.5fM, respectively. The fluorescence values ​​in c, f, and i were determined 50 minutes after incubation, and the data were from ten experiments, each of which was repeated twice. For a, b, d, e, g, and h, each experiment was repeated three times, and a representative result is shown in the figure. For ai, the reaction temperature was 37°C.

[0013] Figure 3Competition between RPA and crRNA / Cas12a RNP cleavage in a one-pot reaction. (ab) Accumulation of RPA amplicons in a one-pot reaction. RPA components, crRNA / Cas12a RNP at a concentration of 33nM, and 2340fM dsDNA substrate were incubated at 37°C for 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 minutes, and the resulting RPA amplicons were analyzed in agarose gels. Arrows indicate amplicon products. (cd) Amplification and depletion of amplicons in a one-pot reaction. Each concentration of 1170fM suboptimal and classic PAM substrates was mixed in a 1:1 ratio in a one-pot reaction at 37°C, and the percentage of each concentration at 0, 1, 3, 5, 7, 10, 15, and 20 minutes was determined by deep sequencing. For each time point, n=5. (ef) In vitro cleavage activity of crRNA4 targeting substrates with suboptimal (GTTG) or classical (TTTG) PAM and crRNA5 targeting substrates with suboptimal (CTTA) or classical (TTTA) PAM. The 50nM crRNA / LbCas12a complex was incubated with dsDNA substrates (spacer 4 was 6nM and spacer 5 was 10nM) at 37°C for 0, 0.5, 1, 2, 5, 10, 15 or 20 minutes to determine the cis-cleavage activity. S, substrate; P, product. (gh) The binding affinity of RNP to suboptimal and classical PAM dsDNA was determined. 0, 12.5, 25, 50, 100, 200 and 400nM crRNA / inactivated LbCas12a (dCas12a) complexes were incubated with 5nM dsDNA at 37°C for 20 minutes, and EMSA was performed to determine the bound and unbound fractions. Each experiment was repeated three times, and one representative is shown in the figure.

[0014] Figure 4 Cis-cleavage activity of 120 PAMs for four targets. (a) Correlation of one-pot reactions of 120 PAMs with cis-cleavage. Black dots represent classic PAMs, red dots represent suboptimal PAMs with better performance, and blue dots represent suboptimal PAMs with worse performance. The unit (X-axis) of the one-pot reaction is defined as the time to reach half-maximal fluorescence (min)*adjusted ratio based on the platform signal of each PAM. The ratio is the highest platform fluorescence value among the 120 PAMs divided by the platform fluorescence value of each PAM. The three suboptimal PAMs outside the 30min range of the X-axis still perform better than their corresponding classic PAMs. (b) Schematic workflow of competition between amplification and cleavage in a one-pot reaction. For substrates containing classic PAMs, cleavage dominates in the initial stage of the reaction, resulting in excessive consumption of dsDNA activators. In contrast, since amplification of suboptimal PAM substrates outperforms cleavage, amplicons accumulate to stimulate faster and stronger fluorescence signal generation.

[0015] Figure 5 Detection of HCMV by suboptimal PAM-mediated one-pot reaction. (ad) Comparison of the sensitivity of the suboptimal PAM-mediated one-pot reaction and qPCR assay targeting the UL55 gene of HCMV. (ab) PUC57-UL55 plasmid was used as substrate. (cd) HCMV virus was substrate. The reaction volume of qPCR in a and c was 20 μL, while the reaction volume of the one-pot reaction in b and d was 30 μL, and the copy number input in both reactions was the same. (e) Schematic diagram of detection under portable UV light and using lateral flow strips. (f) Visualization of direct fluorescence stimulated by UV light for detection of HCMV virus. The reaction was checked under UV light at 8, 10, 15 and 20 minutes after incubation at 37°C. (g) Twenty minutes after incubation, the lateral flow strip was immersed in the reaction tube for 5 minutes to visualize the control band and the test band.

[0016] Figure 6 Detection of SARS-CoV-2 using a suboptimal PAM-mediated one-pot reaction. (a) Genomic maps of classical PAM (TTTV) and suboptimal PAM (VTTV, TTVV, TCTV) spacers in SARS-CoV-2. (b) Detection limits of DNA and RNA by FASTER (b, d) at 42°C and STOPCovid.v1 (c, e) at 60°C. The number of molecules input into FASTER and STOPCovid.v1 was the same. (f) FASTER results for 204 SARS-CoV-2 nasopharyngeal swab samples obtained from patients (left: 104 positive samples, 48 ​​unextracted samples marked with solid circles, and 56 pre-extracted samples marked with open circles; right: 100 negative samples). Fluorescence readings were measured at 42°C at 20 minutes. The threshold was determined as three times the average of the initial fluorescence values ​​of all samples, S / N (signal-to-noise ratio) = 3. (g) Direct visualization of unextracted SARS-CoV-2 positive samples under UV light. Reactions were checked under UV light after 10, 15 and 20 min of incubation at 42° C. (h) Summary table between FASTER and RT-qPCR for 204 samples.

[0017] Figure 7 Suboptimal and classical PAM-mediated one-pot assay. One-pot assay using spacers with suboptimal or classical PAMs in the Orf1ab (a) and E (b) genes of SARS-CoV-2. crRNA1-3 targeting the Orf1ab gene and crRNA2-7 targeting the E gene used classical PAMs, while crRNA4-5 targeting the Orf1ab gene and crRNA1 targeting the E gene used suboptimal PAMs. (c) PAMs and spacers used in the one-pot reaction.

[0018] Figure 8 Comparison of the incidental activity and one-pot reaction of various suboptimal and classical PAMs. (ad) Summary of the fluorescence kinetics of the suboptimal PAMs and three classical PAMs with point mutations at positions 1-3 in the incidental activity assays (a and b) and the corresponding one-pot reactions of HPV18 L1 spacer 1 (c) and SARS-CoV-2 S spacer 2 (d). Determination of the time to reach half-maximal fluorescence. Incidental activity and fluorescence values ​​of the one-pot reactions determined at 40 min and 20 min, respectively. 2.7 nM and 2.8 nM dsDNA substrate were added to the incidental activity assays of HPV18 L1 spacer 1 and S spacer 2; 18.3 fM and 189 fM dsDNA substrate were added to the one-pot reaction assays of HPV18 L1 spacer 1 and S spacer 2, and these assays were performed at 37 °C, n = 3. Fluorescence detection of Orf1ab spacer 5 in the incidental activity. The CTTA PAM was mutated to TTTA, TTTG, and TTTC, and T1-T3 in the TTTV PAM were mutated to A, G, and C, respectively. PAMs based on the T1-T3 mutations of the TTTA, TTTG, and TTTC PAMs were determined to compare the incidental activities.

[0019] Fig. 9 Spontaneous activity and one-pot reaction of Orf1ab spacer 4 using various PAMs. GTTG PAM was mutated to TTTA, TTTG and TTTC as canonical PAM, and T1-T3 in TTTV PAM was mutated to A, G or C, respectively. (ai) Spontaneous activity of suboptimal PAMs mutated to T1-T3 and related canonical PAMs were compared. (jr) One-pot reaction of suboptimal PAMs mutated to T1-T3 and related canonical PAMs were compared. 3.5 nM dsDNA was added to the spin-off activity assay, and 2.3 pM dsDNA was added to the one-pot reaction assay, and these assays were performed at 37°C, n=3.

[0020] Fig.10 Spontaneous activity and one-pot reaction of Orf1ab spacer 5 using various PAMs. CTTA PAM was mutated to TTTA, TTTG and TTTC as canonical PAM, and T1-T3 in TTTV PAM was mutated to A, G or C, respectively. (ai) Spontaneous activity of suboptimal PAMs with T1-T3 mutations and related canonical PAMs were compared. (jr) One-pot reaction of suboptimal PAMs with T1-T3 mutations and related canonical PAMs were compared. 3.5 nM dsDNA was added to the spin-off activity assay, and 2.3 pM dsDNA was added to the one-pot reaction assay, and these assays were performed at 37°C, n=3.

[0021] Fig.11The incidental activity and one-pot reaction of HPV18 L1 gene spacer 1 using various PAMs. The TTAC PAM was mutated to TTTA, TTTG, and TTTC as the classical PAM, and T1-T3 in the TTTV PAM was mutated to A, G, or C, respectively. (ai) Comparison of the incidental activity of the suboptimal PAMs with T1-T3 mutations and the relevant classical PAMs. (jr) Comparison of the one-pot reaction of the suboptimal PAMs with T1-T3 mutations and the relevant classical PAMs. 2.7nM dsDNA was added to the incidental activity assay, and 18.3fM dsDNA was added to the one-pot reaction assay, which were performed at 37°C, n=3. Fluorescence detection of Orf1ab spacer 5 in one-pot reaction.

[0022] Fig.12 Spontaneous activity and one-pot reaction of S-gene spacer 2 using various PAMs. TTCT PAM was mutated to TTTA, TTTG and TTTC as canonical PAMs, and T1-T3 in TTTV PAM was mutated to A, G or C, respectively. (ai) Spontaneous activity of suboptimal PAMs mutated to T1-T3 and related canonical PAMs were compared. (jr) One-pot reaction of suboptimal PAMs mutated to T1-T3 and related canonical PAMs were compared. 2.8 nM dsDNA was added to the spin-off activity assay, and 189 fM dsDNA was added to the one-pot reaction assay, which were performed at 37°C, n=3.

[0023] Fig.13 Schematic diagram of Cas12a recognizing and interacting with the PAM duplex. A paired with T2 directly forms hydrogen bonds with the conserved Lys538 and Lys595 of Cas12a (modified from Yamano T, Mol Cell, 2017).

[0024] Fig.14 PAM-mediated incidental activity and one-pot reactions of two- and three-point mutations. (ad) Incidental activity (a, c) and one-pot reactions (b, d) of Orf1ab spacer 4 and 5 targeting with TTNT PAM. (ef) Incidental activity (e) and one-pot reactions (f) of Orf1ab spacer 4 targeting with VVTV, VTVV PAM. (gh) Incidental activity (g) and one-pot reactions (h) of Orf1ab spacer 5 targeting with VVTV, VTVV, and TCCV PAM. (ij) Incidental activity (i) and one-pot reactions (j) of Orf1ab spacer 5 targeting with CCCV and AGCV PAM. 3.5 nM dsDNA was added to the incidental activity assays, and 2.3 pM dsDNA was added to the one-pot reaction assays, which were performed at 37°C, n=3.

[0025] Fig.15 Comparison of suboptimal and classical PAM-mediated one-pot reactions. E spacer 8 (a) and S spacer 3 (b) of SARS-CoV-2 were examined. The concentrations of dsDNA in the one-pot reactions were 325.5 fM and 189 fM for E spacer 8 and S spacer 3, respectively. The one-pot reactions were performed at 37°C.

[0026] Fig.16 Determination of the dose effect of RNP in one-pot assays. RNP doses ranging from 5.5, 11, 22, 33, 66 to 132 nM were tested in one-pot reactions using suboptimal PAM (a) and classical PAM (b) at 37°C. dsDNA was added to the one-pot reactions at a concentration of 2.3 pM.

[0027] Fig.17 The amount of RPA amplicons accumulated in a one-pot reaction. The components of RPA, crRNA / Cas12a RNP, and dsDNA substrate were incubated at 37°C for 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 minutes, and the RPA amplicons were analyzed in agarose gel. RPA alone represents a one-pot reaction in the absence of crRNA / Cas12a RNP; TTTG, TTTC and TTCG, TTAC represent one-pot reactions using classical PAM and suboptimal PAM, respectively. Arrows indicate RPA amplicons.

[0028] Fig.18 Cis-cleavage activity of 120 PAMs for four targets. In vitro cleavage activity of dsDNA substrates containing HPV18 L1 gene spacer 1, Orf1ab spacer 4, Orf1ab spacer 5, and S gene spacer 2 with suboptimal PAMs (VTTV, TVTV, TTVV) and classical PAMs (TTTV). RNPs and dsDNA were incubated at 37°C for 0, 1, 5, 10, or 20 minutes. The dsDNA substrates for HPV18 L1 gene spacer 1, Orf1ab spacer 4, Orf1ab spacer 5, and S gene spacer 2 were 7.5 nM, 11 nM, 6 nM, and 9 nM, respectively. HPV18 L1 gene spacer 1, S: 591bp, P: 382bp, 209bp; Orf1ab spacer 4, S: 539bp, P: 388bp, 151bp; Orf1ab spacer 5, S: 461bp, P: 220bp, 241bp; S gene spacer 2, S: 570bp, P: 257bp, 313bp.

[0029] Fig.19Comparison of one-pot reactions based on AMP future and TwistDx. (a) Comparison of amplification using AMP future and TwistDx kits. RPA amplification was performed at 42°C for 20 minutes. (b) Fluorescence comparison of one-pot reactions based on AMP future and TwistDx. 8403aM, 840.3aM, 84.03aM, 8.403aM dsDNA were applied. (c) RNP dosage optimization in one-pot reactions based on TwistDx. 333, 200, 100 or 33.3nM RNP were applied. 8403aM DNA substrate was used. Suboptimal PAM was used in bc. (dg) Comparison of suboptimal and classical PAM-mediated one-pot reactions. Suboptimal PAMs and classical PAMs were compared for SARS-CoV-2 Orf1ab spacer 4, Orf1ab spacer 5, E gene spacer 8, and HPV18 L1 gene spacer 1 using the TwistDx kit and 100 nM RNP. The input dsDNA substrate concentrations were 2340 fM for Orf1ab spacer 4 and spacer 5, 325.5 fM for E gene spacer 8, and 243.9 fM for HPV18 L1 gene spacer 1.

[0030] Fig. 20 Number of spacers with classical and suboptimal PAMs counted in HCMV and SARS-CoV-2. (a) Spacers with classical (TTTA, TTTG, TTTC) and suboptimal PAMs (VTTV, TTVV, TCTV) in HCMV. (bc) Classical and alternative suboptimal PAMs (A-suboptimal PAM, TTNT, TRTV, YYYN (except TTTV)) in SARS-CoV-2 (b) and HCMV (c).

[0031] Fig.21 Optimization of reverse transcription reaction. (a) One-pot reaction was performed at 42°C, 43°C, and 45°C. (b) Screening of reverse transcriptase with efficient primers. RT products were quantified by qPCR after the RT enzyme reacted at 48°C for 30 min. (cd) UV images and fluorescence of FASTER in the presence of different concentrations of RPA reverse primer (RPA-R) and reverse transcription primer 1 (RT-1) using extracted virus samples. NC represents the reaction in the absence of substrate.

[0032] Fig. 22 Limit of detection (LOD) of RT-qPCR for SARS-CoV-2 virus-like particles. (a) LOD of RT-qPCR using CDC N2 primer pair. Input substrate is 10 4 , 10 3 , 10 2 , 101 , 10 0.5 , 10 0 and 10 -0.5 Copies / μL. (b) CDC RT-qPCR assay standard curve. Ten-fold dilutions of input substrate were used to generate the standard curve, and each dilution was repeated three times. (c) FASTER detection limit for SARS-CoV-2 virus-like particles.

[0033] Fig.23 FASTER performed on patient samples. (ab) Positive NP swabs detected by UV light imaging. (a) Unextracted sample and (b) extracted sample. UV images captured at 15-20 min. (c) Unextracted NP swabs with different Ct values ​​imaged by UV light and simple blue light device. (Left: 0 min, right: 20 min)

[0034] Fig.24 STOPCovid, version 1 (STOPCovid.v1) performed on patient samples. (ab) 104 positive patient samples and 19 negative patient samples were detected by STOPCovid.v1. 48 unextracted samples were marked with solid circles and 56 extracted samples were marked with open circles. Fluorescence values ​​were read at 45 min. (c) Results evaluation of STOPCovid.v1 and RT-qPCR.

[0035] Fig.25 Specificity evaluation of FASTER. (a) Alignment of the SARS-CoV-2 N gene target region with the common human coronavirus N genes (including MERS, HKU1, 229E, NL63, and OC43); (b) Identification of amplicons by RPA assay with 1E5 copies / each coronavirus N gene dsDNA in a 15-min reaction at 37°C; arrows represent amplicons. (c) Fluorescence kinetics of incidental activity tested at 37°C using 2.5 nM dsDNA input of different sequences; (d) Fluorescence kinetics of one-pot reactions tested at 42°C using 1E5 copies / each coronavirus N gene RNA.

[0036] Fig.26 Comparison of CRISPR-based SARS-CoV-2 detection methods. The substrates used to evaluate sensitivity were as follows: SARS-CoV-2 virus-like particles for FASTER, N gene RNA for DETECTR and amplification-free detection, extracted genomic RNA for SHERLOCK and SHINE, SARS-CoV-2 genomic standards for STOPCovid.v1, and concentrated samples for STOPCovid.v2.

[0037] Fig. 27 LbCas12a mutants mediate faster one-pot reactions than wild-type proteins. (ag) PAM-related residues 595K and 595K&542Y of LbCas12a protein were mutated to alanine. One-pot reactions of LbCas12a WT and mutants on Orf1ab gene (ab), E gene (cd) and N gene (eg). Reactions were performed at 42°C, with 20pg dsDNA substrate used for ad and various doses used for eg.

[0038] Fig.28 AapCas12b mutants mediate faster one-pot reactions than wild-type proteins. (a) Cis-cleavage of AapCas12bWT and 3M on 100ng N gene dsDNA. NC means reaction in the absence of protein. S is the substrate, and P1 and P2 are the products of cleavage. (b) Trans-cleavage activity of WT and 3M. (c) One-pot reaction combining RPA and AapCas12b. The reaction was performed at 42°C. (de) StopCovid.v1 reaction of WT and 3M at 60°C. (f) Fluorescence value of StopCovid.v1 reaction at 25min. The 3M mutant refers to G478A / K396A / Q403A of AapCas12b. DETAILED DESCRIPTION One-step detection of nucleic acids

[0039] In one embodiment of the present disclosure, a one-step, rapid, sensitive, reliable and flexible method for detecting nucleic acids is provided. The method shows a detection limit comparable to quantitative PCR (qPCR), but the time is significantly shortened, for example from 15 to 20 minutes.

[0040] It has been found that when reagents for isothermal amplification and CRISPR detection are combined to achieve one-step detection, CRISPR proteases can prematurely cleave substrate nucleic acids used as amplification templates. This can result in insufficient or slow amplification, resulting in missed detection or inefficient detection. In some embodiments of the present disclosure, systems and methods for coordinating amplification and CRISPR detection processes are provided so that amplification can be performed efficiently, thereby allowing rapid and efficient detection of amplified products.

[0041] Therefore, according to one embodiment of the present disclosure, a method for detecting a target polynucleotide is provided, the method comprising incubating the target polynucleotide in a mixture comprising (a) a polymerase, (b) deoxynucleoside triphosphates (dNTPs), (c) primers for amplifying the target polynucleotide, (d) a CRISPR-associated (Cas) nuclease, and (d) a guide RNA comprising a spacer segment complementary to a target segment on the target polynucleotide.

[0042] In some embodiments, polymerase, primers and dNTPs can isothermally amplify target polynucleotides.Isothermal amplification techniques are well known in the art.Isothermal amplification methods provide detection of nucleic acid target sequences in a streamlined, exponential manner and are not limited by thermal cycling.Although these methods can be very different, they all share some common features.For example, because DNA chains do not have thermal denaturation, all isothermal methods rely on an alternative method to achieve the initiation of primer binding and amplification reactions: a polymerase with strand displacement activity.Once the reaction is initiated, the polymerase must also separate the chain that is still annealed to the target sequence.

[0043] Isothermal methods usually use unique DNA polymerases to separate double-stranded DNA. DNA polymerases with this ability include Klenow exo-, Bsu large fragment and EquiPhi29, phi29 for moderate temperature reactions (25°C-40°C), and Bst, Bsm DNA large fragment polymerases for higher temperature reactions (50°C-65°C). To detect RNA species, a reverse transcriptase compatible with the reaction temperature is added (except for NASBA / TMA reactions) to maintain the isothermal nature of the amplification.

[0044] An example of isothermal amplification is loop-mediated isothermal amplification (LAMP). LAMP uses 4-6 primers that recognize 6-8 different regions of the target DNA. Strand displacement DNA polymerase initiates synthesis, and 2 primers form a loop structure to promote subsequent rounds of amplification. LAMP is fast, sensitive, and amplifies so extensively that the magnesium pyrophosphate produced during the reaction is visible to the naked eye, making LAMP very suitable for field diagnosis.

[0045] Another example of isothermal amplification is whole genome amplification (WGA). WGA is a multiple displacement amplification (MDA) method that uses the strand displacement activity of DNA polymerases (such as EquiPhi29, phi29 or Bst, Bsm DNA polymerases) to achieve robust amplification of the entire genome. WGA has become an invaluable method for limited samples of precious stock materials or to achieve single-cell genomic DNA sequencing. The reaction products are very long (>30kb) and highly branched through multiple displacement mechanisms.

[0046] Another example of isothermal amplification is strand displacement amplification (SDA). SDA or a similar method (nicking enzyme amplification reaction (NEAR)) relies on a strand-displacing DNA polymerase, usually Bst DNA polymerase, large fragment or Klenow fragment (3'-5' exo-), to initiate at a nick made by a strand-restricted restriction endonuclease or nickase at a site contained in the primer. Each polymerase displacement step regenerates the nicking site, resulting in exponential amplification. NEAR is very rapid and sensitive and can detect small amounts of target in a few minutes.

[0047] Another example of isothermal amplification is helicase-dependent amplification (HDA). HDA uses the double-stranded DNA unwinding activity of a helicase to separate the strands, thereby enabling primer annealing and extension by a strand-displacing DNA polymerase. Like PCR, this system requires only two primers.

[0048] Another example of isothermal amplification is recombinase polymerase amplification (RPA). RPA uses recombinase to help primers invade double-stranded DNA. T4 UvsX, UvsY and the single-strand binding protein T4 gp32 form D-loop recombination structures that initiate amplification by strand-displacing DNA polymerases. RPA is typically performed at about 37°C-42°C and, unlike other methods, can produce discrete amplicons up to 1 kb.

[0049] Another example of isothermal amplification is nucleic acid sequence-based amplification (NASBA). NASBA and transcription-mediated amplification (TMA) are both isothermal amplification methods performed with RNA. Primers are designed to target the region of interest; one of the primers must contain the promoter sequence for T7 RNA polymerase at the 5' end. There are other isothermal amplifications, including rolling circle amplification (RCA), asymmetric isothermal amplification (SMAP 2), exponential amplification reaction (EXPAR), beacon-assisted detection amplification, single primer isothermal amplification (SPIA), and cross-primer amplification (CPA).

[0050] In some embodiments, one or more components or conditions are adjusted so that the polymerase can effectively amplify the target polynucleotide and the Cas nuclease can cleave the amplified target polynucleotide. In one embodiment, the target polynucleotide is amplified by at least 10% in 10 minutes. 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 fold amplification, while the Cas nuclease and guide RNA are present in the mixture.

[0051] This can be achieved in a variety of ways. In one example, the binding between the Cas nuclease (or Cas protein) and the guide RNA or between the Cas-guide RNA ribonucleoprotein (RNP) and the target polynucleotide is reduced. In another example, the cutting efficiency of the Cas protein is reduced. A. Suboptimal PAM

[0052] An example method to reduce the binding between RNP and target polynucleotide is to design guide RNA to target suboptimal PAM. The protospacer adjacent motif (PAM) is a 2-8 base pair DNA sequence that follows the sequence targeted by the Cas nuclease in the CRISPR bacterial adaptive immune system. PAM is an important targeting component. Each Cas nuclease has one or more classical PAM sequences, as well as some non-classical sequences. Non-classical PAMs are not optimal and may result in lower efficiency of binding and cutting.

[0053] In one embodiment, the guide RNA is designed such that it includes or is adjacent to a protospacer adjacent motif (PAM) sequence that can be recognized by the Cas nuclease and is suboptimal or non-canonical.

[0054] For each known Cas nuclease, the corresponding classical and non-classical PAMs are known. For example, for LbCas12a, non-classical PAM sequences include NTTV, TNTV, TTNV (except TTTV), TTNT, VTTT, TVTT, VVTT, VTVT, VNVV, NVNV, NVVV, VNTV, NTVV, TNVV and VVNV, YYYN, where N represents any nucleotide. For AapCas12b, non-classical PAM sequences are VTN, TTN (except TTV), TVN, NVN and VVN, where N represents any nucleotide. B. Modified Cleavage Substrates

[0055] Certain modifications to the target polynucleotides of the CRISPR system may reduce binding affinity and / or cleavage efficiency without affecting amplification. Therefore, by incorporating these modifications into the substrate, Cas cleavage may be inhibited or delayed, thereby allowing sufficient amplification.

[0056] Such modifications can be incorporated into the amplified target polynucleotide via modified dNTPs and / or modified primers.

[0057] In some embodiments, dNTP is substituted with an analog or variant, such as deoxyuridine triphosphate, deoxyinosine triphosphate, pseudouridine triphosphate, methyl pseudouridine triphosphate, 2-aminopurine, 5-bromo-dU or ribonucleoside triphosphate (rNTP). In some embodiments, dNTP, rNTP or any analog or variant can be modified. Non-limiting examples of modification include those with the following groups, such as phosphoryl, biotin, digoxin, amino, thiol, phosphorothioate and methyl.

[0058] In some embodiments, one or more nucleotides in one or more primers are replaced with analogs or variants, such as deoxyuridine triphosphate, deoxyinosine triphosphate, pseudouridine triphosphate, methyl pseudouridine triphosphate, 2-aminopurine, 5-bromo-dU or ribonucleoside triphosphate (rNTP). In some embodiments, nucleotides, rNTPs or any analogs or variants can be modified. Non-limiting examples of modifications include those with the following groups, such as phosphoryl, biotin, digoxin, amino, thiol, phosphorothioate and methyl.

[0059] The percentage of such modified nucleotides can be adjusted as needed. A higher percentage of modification can reduce CRISPR binding / cutting efficiency more, and vice versa. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20% of the dNTPs or nucleotides in the primers are modified. In some embodiments, no more than 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% or 60% or 70% of the dNTPs or nucleotides in the primers are modified.

[0060] The primers of the reaction can be chemically modified to improve isothermal amplification, or in some embodiments, the primers have a partial sequence of the crRNA spacer or have a partial or complete suboptimal PAM sequence, and the primers can be chemically modified or not. C. Modified guide RNA

[0061] In another example, compared to the standard guide RNA structure, the guide RNA (or crRNA) is modified to inhibit the formation of RNP or the binding between RNP and the target nucleotide. Examples of modified guide RNA / crRNA are provided below.

[0062] In some embodiments, crRNA is truncated at the 3' end of the region complementary to the target nucleic acid. In some embodiments, the truncated crRNA contains 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or even less nucleotides complementary to the target polynucleotide.

[0063] In some embodiments, the crRNA is truncated at the 5' end of the hairpin region. In some embodiments, the guide RNA includes a truncated tracrRNA sequence. In some embodiments, the truncation is 1, 2, 3, 4 or 5 nucleotides.

[0064] In some embodiments, the hairpin structure of crRNA is extended, for example, at the 3' end of the spacer (e.g., 5'-AGACAUGGACCA-3'). In some embodiments, the stem region includes an extended sequence. In some embodiments, the loop region includes an extended sequence. The extension is at least 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 nucleotides. The length of the hairpin sequence can be 1 to 100nt or even longer.

[0065] In some embodiments, the crRNA or tracrRNA is extended at the 5' end and / or the 3' end, for example, at the 3' end of the spacer (e.g., 5'-AGACAUGGACCA-3'). The extension is at least 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 nucleotides. The length of the sequence can be 1 to 100 nt or even longer, and any desired modification can be incorporated.

[0066] In some embodiments, the nucleotides of the guide RNA that interact with the Cas protein through the 2' hydroxyl group of the ribose are replaced by DNA. In some embodiments, the nucleotides at the 5' end of the spacer (1-12nt, longer or the entire sequence) can be modified, whether continuous or discontinuous.

[0067] In some embodiments, one or more nucleotides in the guide region of the guide RNA incorporate one or more locked nucleic acids (LNA) or bridging nucleic acids (BNA). In some embodiments, the one or more nucleotides are located at positions 1-12nt or 12-20nt within the guide region.

[0068] Other exemplary modifications to nucleotides in the guide RNA include deoxynucleotides, locked nucleic acids (LNA), bridged nucleic acids (BNA), deoxyuridine, deoxyinosine, pseudouridine, methylpseudouridine, or modified nucleotides with groups such as phosphoryl, biotin, digoxigenin, amino, thiol, phosphorothioate, methyl, 2'-O-methyl-3'-phosphonoacetate (MP), 2'O-methoxy (MOE), fluoro (F), S-constrained ethyl, 2'-O-methyl-PS (MS), and 2'-O-methyl-thio PACE (MSP).

[0069] In some embodiments, the guide RNA comprises 1, 2, 3, 4, 5 or 6 mismatches in the complementary region (spacer) to the target polynucleotide. The mismatches can be continuous or discontinuous. D.Cas nuclease

[0070] In another embodiment, an engineered Cas nuclease is used that has reduced binding to the guide RNA and / or target polynucleotide, or reduced cleavage activity.

[0071] The term "Cas nuclease", "Cas protein" or "Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein" refers to an RNA-guided DNA or RNA endonuclease associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immune system in Streptococcus pyogenes and other bacteria. Non-limiting examples of Cas proteins include Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Acidaminococcus sp. Cas12a (AsCpf1), Lachnospiraceae bacterium Cas12a (LbCpf1), Francisella novicida Cas12a (FnCpf1). Additional examples are provided in Komor et al., “CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes,” Cell. 2017 Jan 12;168(1-2):20-36.

[0072] Non-limiting examples of Cas nucleases include Cas12a, Cas12b, Cas12c, Cas12d, Cas12e (CasX), Cas12f, Cas12k, Cas13a, Cas13b, Cas13c, Cas13d, Csm6, Csm3, and Cas14a, Cas14b, and Cas14c.More specific examples include AsCas12a, FnCas12a, MbCas12a, Lb3Cas12a, Lb2Cas12a, BpCas12a, PeCas12a, PbCas12a, SsCas12a, CMtCas12a, EeCas12a, LiCas12a, PcCas12a, PdCas12a, PmCas12a, ArCas12a, HkCas12a, ErCas12a, BsCas12a, LpCas12a, PrCas12a and PxCas12a (belonging to the Cas12a class), AapCas12b, AmCas12a 2b, AacCas12b, BsCas12b, BvCas12b, BthCas12b, BhCas12b, AkCas12b, EbCas12b, LsCas12b (belonging to the Cas12b class), Mi1Cas12f2, Mi2Cas12f2, Un1Cas12f1, Un2Cas12f1, AuCas12f2, PtCas12f1, AsCas12f1, RuCas12f1, SpCas12f1, and CnCas12f1 (belonging to the Cas12f class (Cas14a)), ShCas12k (CAST) and AcCas12k (belonging to the in the Cas12k class), LwaCas13a, LbaCas13a, LshCas13a, PprCas13a, EreCas13a, LneCa3a, CamCas13a, RcaCas13a, HheCas13a, LbuCas13a, LseCas13a, LbmCas13a, LbnCas13a, RcsCas13a, RcrCas13a, RcdCas13a, CgCas13a, Cg2Cas13a, LweCas13a, LbfCas13a, Lba4Cas13a, Lba9Cas13a, LneCas1 3a, HheCas13a and RcaCas13a (belonging to the Cas13a class), BzCas13b, PbCas13b, PspCas13b, RanCas13b, PguCas13b, PsmCas13b, CcaCas13b, AspCas13b, PauCas13b, Pin2Cas13b and Pin3Cas13b (belonging to the Cas13b class), RspCas13d, RfxCas13d, EsCas13d and AdmCas13d (belonging to the Cas13d class), and TtCsm6, EiCsm6 and LsCsm6 (belonging to the Csm6 class).

[0073] Exemplary mutations that reduce binding to the guide RNA and / or target polynucleotide or reduce the cleavage activity of the Cas nuclease are provided in the table below.

[0074] Based on structural analysis, the residues in Table A are involved in the formation of ribonucleoprotein (RNP). Table A. Residues that affect RNP formation

[0075] The residues in Table B are considered important for maintaining the conformation of the Cas nuclease. Table B. Residues important for Cas conformation

[0076] The residues in Table C are believed to be involved in the interaction with the PAM sequence. Table C. Residues interacting with PAM

[0077] Conserved amino acids in the REC1 (24-282) and REC2 (283-521) domains and the helix I (14-391) and helix II (660-822) domains are thought to be important for the structure or activity of Cas nucleases. Another example is the residues in the HNH domain.

[0078] In addition, residues on the REC lobe, Nuc lobe or RuvC domain that form hydrogen bonds with the target DNA can also be targeted for mutation. Other suitable mutation targets are positively charged residues or negatively charged residues. Examples are provided in Table D below. Table D. Residues that interact with target DNA or RNA

[0079] In some embodiments, the amino acid residues identified above can be deleted to be substituted by different amino acids.In some embodiments, the substitution is a non-conservative substitution.

[0080] Whether a substitution is a non-conservative substitution can be determined using well-known knowledge, for example, using the matrix in Table E below. In Table E, a negative similarity score indicates a non-conservative substitution between two amino acids. Table E. Amino Acid Similarity Matrix C G P S A T D E N Q H K R V M I L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 K -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 P -3 -1 6 G -3 5 C 12

[0081] In some embodiments, the substitution is with alanine. E. Adjusted reaction conditions

[0082] In another embodiment, reaction conditions are adjusted to favor amplification rather than CRISPR cleavage.

[0083] In one example, the amount of Cas nuclease / guide RNA in the mixture is adjusted to reduce the cleavage efficiency. In another example, the magnesium ion concentration is increased or decreased to reduce the cleavage efficiency or increase the amplification efficiency.

[0084] In another example, a certain amount of dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), tween 20, proteinase K inhibitor or nuclease inhibitor is added to the reaction system. In another example, the pH of the reaction mixture is adjusted to, for example, between 5.0 and 10.0. In another example, the reaction temperature is adjusted.

[0085] In yet another example, one or more additives (see examples in Table F) are added to the reaction mixture to reduce binding between the RNP and the target polynucleotide. Table F. Example Additives

[0086] Using any one or combination of these methods, the target polynucleotide can be fully amplified, followed by guide RNA-directed cleavage by the Cas nuclease. The cleaved target polynucleotide can then be detected using a variety of different techniques known in the art.

[0087] For example, a cleavage event can be detected using a toehold switch sensor that can generate a colorimetric output on a test strip. Toehold switches are synthetic RNAs that mimic messenger RNAs, whose job is to transfer information from DNA to the protein synthesis machinery. They contain a recognition sequence (toehold) for a specific stimulus in the form of a specific "input" RNA, as well as a recognition sequence that the protein synthesis machinery (ribosome) needs to bind to in order to initiate translation of the fused protein coding sequence into its encoded protein product. In the absence of "input" RNA, the toehold switch is maintained in its closed (OFF) state by forming a hairpin structure that uses a partial "input" recognition sequence and a ribosome recognition sequence (remaining inaccessible). When the stimulated "input" RNA binds to the toehold and induces the hairpin structure to open, the toehold switch is turned on, enabling the ribosome to access its recognition sequence to begin synthesis of downstream encoded proteins, thereby generating a detectable signal.

[0088] In another example, a quenched fluorophore is added to the substrate, and once the substrate is cleaved, the fluorophore is released and thus emits fluorescence, thereby achieving target detection.

[0089] The methods herein can be used to detect or quantify different types of nucleic acids, such as single-stranded RNA, double-stranded RNA, single-stranded DNA or double-stranded DNA. The nucleic acids can be from any type of sample, such as a clinical sample suspected of infection, or a sample requiring mutation or SNP (single nucleotide polymorphism) detection, but are not limited thereto. Compositions and kits for performing these methods

[0090] Also provided are compositions and kits useful for performing the methods of the disclosure.

[0091] In one embodiment, a kit, package or composition for detecting a target polynucleotide is provided. In some embodiments, the kit, package or composition includes (a) a polymerase, (b) deoxynucleoside triphosphates (dNTPs), (c) primers for amplifying the target polynucleotide, (c) CRISPR-related (Cas) nucleases, and (d) guide RNAs comprising spacer fragments complementary to the target fragments on the target polynucleotide. The presence of these components allows the polymerase to effectively amplify the target polynucleotide, while the Cas nuclease is able to cut the amplified target polynucleotide. In some embodiments, the target fragment amplified by the polymerase includes a suboptimal or non-classical PAM sequence targeted by the guide RNA and the Cas nuclease.

[0092] In another embodiment, the kit, package or composition includes (a) a polymerase, (b) deoxynucleoside triphosphates (dNTPs), and (c) primers for amplifying the target polynucleotide, wherein the dNTPs and / or primers are modified / substituted such that the amplified products are reduced in binding to a CRISPR system (such as those described herein). In some embodiments, one or more primers include a PAM sequence for a Cas nuclease. In some embodiments, the PAM sequence is a suboptimal or non-classical PAM sequence.

[0093] Another embodiment provides a kit, package or composition for cleaving a target polynucleotide, the kit, package or composition comprising (a) a CRISPR-associated (Cas) nuclease, and (b) a guide RNA comprising a spacer segment complementary to a target segment on the target polynucleotide, wherein the guide RNA has reduced binding to the target polynucleotide or reduced cleavage of the target polynucleotide compared to a standard guide RNA.

[0094] In another embodiment, a mutant Cas nuclease is also provided, which has (a) reduced activity in forming ribonucleoprotein (RNP), (b) an altered conformation, (c) reduced activity in interacting with a target PAM sequence, or (d) reduced binding to a target polynucleotide to be cleaved.

[0095] In some embodiments, the polymerase is an enzyme capable of amplifying a target polynucleotide together with dNTPs and primers. In some embodiments, the amplification is an isothermal amplification. Example polymerases and corresponding isothermal amplification systems are described above.

[0096] In some embodiments, one or more dNTPs are modified. In some embodiments, the modification results in reduced binding or cleavage of the Cas nuclease. Examples of such modifications are also provided herein. Suitable percentages of such modifications are also described herein.

[0097] In some embodiments, one or more nucleotides in one or more primers are modified. In some embodiments, the modification results in reduced binding or cutting of the Cas nuclease. Examples of such modifications are also provided herein. Suitable percentages of such modifications are also described herein.

[0098] In some embodiments, primers and / or guide RNAs are designed such that a suboptimal PAM sequence is included in the amplified sequence for targeting by the guide RNA / Cas nuclease. Examples of such suboptimal PAM sequences and their corresponding Cas nucleases are also described herein.

[0099] In some embodiments, the guide RNA is designed so that its binding to the Cas nuclease or target polynucleotide is reduced. Such designs include, but are not limited to, truncations, extensions, modifications. Examples are also provided herein.

[0100] In some embodiments, sequence engineered Cas nucleases are provided, which have reduced binding to guide RNA or target polynucleotides, or reduced cleavage of target polynucleotides. Such mutations and exemplary residues of exemplary mutations are also provided in the present disclosure.

[0101] The methods and compositions disclosed herein can be used to quickly and effectively detect nucleic acids, such as clinical samples with potential viral infections, genomic DNA with potential SNPs (single nucleotide polymorphisms), but are not limited thereto. As demonstrated, the present application provides a simple, instrument-free and sensitive alternative to the gold standard PCR, and has great potential for rapid, instant screening of target nucleic acid molecules. DETAILED DESCRIPTION Example 1: Accelerated one-pot detection with sensitivity, reliability and flexibility using suboptimal PAM of Cas12a

[0102] This embodiment demonstrates a flexible, accelerated, suboptimal PAM-based detection, which is a (FASTER) detection method with enhanced sensitivity and reproducibility. This embodiment found that in a one-step CRISPR detection, but the isothermal amplification and cutting of Cas12a occurred simultaneously in the same tube, the crRNA targeting the substrate with a suboptimal PAM instead of the conventionally used classical PAM can speed up the reaction by 2-3 times. In addition, a large number of tests have shown that the FASTER detection has higher sensitivity and reliability due to less interference with the isothermal amplification of Cas12a. The prevalence of suboptimal PAM is much higher, making the development of detection kits more flexible and thus optimized. The FASTER detection allows the detection of the DNA virus human cytomegalovirus in just 8 minutes, and the detection of the RNA virus SARS-CoV-2 in 15 minutes, with detection limits comparable to qPCR in both cases. Due to its fast turnaround time, high sensitivity and reliability, the FASTER detection has great potential in promoting instant diagnosis. Materials and Methods Plasmid and dsDNA preparation

[0103] The envelope (E) and spike (S) genes of SARS-CoV-2 were synthesized and cloned into the pUC57 vector (GenScript Biotech, Nanjing, China). The N gene dsDNA of SARS-CoV-2 was obtained by RT-PCR using inactivated virus, and the N gene dsDNA of other human coronaviruses was synthesized (GenScript Biotech, Nanjing, China). The Orf1ab dsDNA substrate containing the spacer 4 and spacer 5 targeting regions was obtained by PCR. Using inactivated HCMV virus as a template, UL55 dsDNA was obtained by PCR and cloned into the pUC57 vector. The SARS-CoV-2 pseudovirus is a lentivirus packaged with the SARS-CoV-2 N gene (Beyotime Biotechnology, Shanghai, China). Preparation of HCMV

[0104] Virus samples were collected from the supernatant of cells cultured after infection with HCMV. HCMV virus samples were inactivated at 95°C and diluted 1:1 in lysis buffer (QuickExtract DNA extraction solution, Lucigen, USA). The copy number was quantified by qPCR based on a standard curve generated using plasmid DNA. LbCas12a protein expression and purification

[0105] The DNA fragment encoding LbCas12a was cloned into a pET-based expression vector containing a C-terminal 6×His-tag. When the culture density reached an OD of 0.7 600 When, the Escherichia coli strain BL21 (DE3) transformed with the recombinant plasmid was incubated with 0.5mM isopropyl β-D-1-thiogalactoside (IPTG), and it was grown for another 16 hours at 21°C. Protein was purified from cell lysate by Ni-NTA resin and eluted with buffer (20mM Tris-HCl, 500mM NaCl and 500mM imidazole, pH 7.4). Then, gel filtration column (Superdex 200Increase 10 / 300GL) was used to further filter the concentrated protein in the elution buffer containing 20mM Tris-HCl (pH 7.5), 200mM NaCl, and the final storage buffer was composed of 20mM Tris-HCl (pH 7.5), 200mM NaCl, 5% glycerol. RPA and RT-RPA

[0106] One lyophilized RPA pellet was resuspended in 29.4 μL of buffer A, 16.1 μL of nuclease-free water, 1 μL of 20 μM RPA forward primer, and 1 μL of 20 μM RPA reverse primer to form the RPA mixture according to the manufacturer's instructions (Weifang Amp-Future Biotech, Shandong, China). Fig.19 The RPA kit of TwistDx (product code: TABAS03KIT) was used. The RPA mixture of TwistDx was resuspended in 29.5 μL rehydration buffer, 15.6 μL nuclease-free water, 1.2 μL of 20 μM RPA forward primer and 1.2 μL of 20 μM RPA reverse primer. The primer sequences are presented in Table 1. For RT-RPA reactions, 0.9 μL RNase H (50 U / μL stock, New England Biolabs, USA) and 0.45 μL SuperScript IV reverse transcriptase (Thermo Fisher Scientific, USA) or EpiScript RNase H-reverse transcriptase (Lucigen) were added to the RPA mixture (PMID: 32848209; 33219228). The reactions were performed at 37°C or 42°C. In the final version of the RT-RPA reaction, add 1 μL of 5 μM RPA reverse primer and additional RT primer (1 μL of 40 μM) and mix well. Table 1. Primers for RPA and RT-RPA Preparation of crRNA

[0107] The DNA template for in vitro transcription was synthesized by overlapping PCR of two oligonucleotides. One oligonucleotide contained the T7 promoter sequence, while the other contained the spacer sequence. The PCR product was incubated with T7 RNA polymerase at 37°C for 2 h for in vitro transcription. The IVT reaction was treated with DNase I (Promega) at 37°C for 15 min and then purified using the Monarch RNA purification kit (NEB). The sequence of the crRNA is presented in Figure 7 c and Table 2. Table 2. crRNA sequences One-pot assay One-pot assays were performed in 30 μL reaction volumes containing 33 or 100 nM LbCas12a RNP, 400 nM FQ ssDNA reporter (FAM-TTATT-quencher, Takara Biotechnology), dsDNA substrate (Table 3) and RPA or RT-RPA components in plate wells (Corning, USA). RNP complexes, FQ ssDNA reporter (8 μL) and RPA mixture (18 μL) were added to each one-pot reaction well, followed by 2 μL of buffer B and dsDNA activator, and then read by SpectraMax i3x at 37 ° C or 42 ° C. The assay was also monitored under UV light, blue light or by lateral flow detection (Milenia HybriDetect 1 kit, TwistDx, UK). The final concentration of the reporter for UV detection was adjusted to 0.4-2 μM. The reporter for the lateral flow assay was FAM-TTATTATT-biotin at a final concentration of 800 nM. Figure 1 , Figure 8-12 and 14, the concentration of dsDNA substrate used was 18.3 fM-2.3 pM. Table 3. PAM and target sequences Deep sequencing

[0108] The deep sequencing samples were prepared as one-pot detection reactions, except that the substrates were mixed with classic PAM and suboptimal PAM substrates in a 1:1 ratio. The reactions were terminated by adding proteinase K (Thermo Fisher Scientific) at different time points, followed by heating at 95°C for 5 minutes to inactivate the protease. The products were amplified using Illumina NovaSeq adapters and barcodes (Table 4), and the resulting reads were filtered by an average Phred quality (Q score) of at least 25. The raw reads were analyzed by a Python script, and the data were normalized based on the reads at the 0-minute time point. Table 4. Deep sequencing primers Fn and Rn represent a pair of primers. Cas12a cleavage and incidental activity in vitro

[0109] For in vitro cleavage, LbCas12a RNP was incubated in 1×NEBuffer 2.1 at room temperature for 20 minutes and then incubated with dsDNA at 37°C. The reaction was terminated by adding proteinase K at different time points and the products were visualized on a 2% TAE gel. The concentration of RNP used was 50 or 100 nM, and the concentration of dsDNA substrate was 6-7.5 nM or 9-11 nM. The percentage of substrate and product was quantified by Image Lab software (Bio-rad). The cleavage efficiency at each time point was plotted as a function of time, and these data were fitted to a single-phase exponential decay curve to calculate K 切割 Values ​​(Prism 8, GraphPad Software, Inc.) (PMID: 26545076). The incidental activity assay was performed in a 30 μL volume containing 33 nM LbCas12a RNP and 400 nM ssDNA reporter (FAM-TTATT-BHQ1) in 1× NEBuffer 2.1, and the fluorescence signal was recorded by SpectraMax i3x. The concentration of the dsDNA substrate activator used was 2.7-3.5 nM. EMSA

[0110] Inactivated LbCas12a (D832A) (referred to as dCas12a) was expressed and purified as described above. Electrophoretic mobility shift assays were performed with dLbCas12a RNP and 50-nt dsDNA substrate labeled with 5'-FAM using 1×NEBuffer2.1. Binding was performed at 37°C for 15 minutes, and then 5% glycerol was supplemented to the reaction. The samples were then resolved on a 4% Tris-borate / EDTA polyacrylamide gel at 120V for 15-20 minutes, and the results were visualized by a fluorescent image analyzer. qPCR and RT-qPCR assays

[0111] qPCR assays for HCMV samples were performed in a 20 μL reaction volume containing 10 μL of 2×AceQ qPCR probe master mix (Vazyme, Nanjing, China), 1 μL of 10 μM of each primer pair (Table 5), and 0.2 μL of 10 μM TaqMan probe (GenScript, China). The number of virus copies input and sample processing in qPCR and FASTER was the same. Each RT-qPCR reaction for SARS-CoV-2 samples contained 10 μL of 2×One Step SYBR Green mix, 1 μL of One Step SYBR Green enzyme mix (Vazyme, China), and 0.4 μL of 10 μM primer pairs. The input volume for RT-qPCR assays was 1.34 μL sample / 20 μL reaction. Table 5. qPCR primers, probe sequences, PCR primers and RT primers SARS-CoV-2 clinical sample collection

[0112] The clinical samples used in this study were approved by the Wuhan Jinyintan Hospital Ethics Committee (KY-2021-01.01). SARS-CoV-2 positive and negative samples were obtained from Wuhan Jinyintan Hospital. Unextracted samples were lysed at 95°C for 5-10 minutes with an equal volume of lysis buffer containing 1U / μL RNasin Plus, 250μM TCEP, and 0.02μg / μL Chelex-100 (PMID: 32577657; 32958655). The extracted RNA samples were purified according to the manufacturer's protocol (Liferiver, Shanghai). They were mixed with RT primers for FASTER detection. UV images of all samples were processed in Image Lab (Bio-Rad) under the following parameters: exposure time: 0.368-0.636, γ value: 0.9-1.14. The STOPCovid.v1 assay was performed exactly as per the protocol (PMID: 32937062). result

[0113] Here, we describe a rapid and simple CRISPR-based diagnostic for the detection of DNA and RNA viruses, including SARS-CoV-2. First, we designed a number of crRNAs targeting the ORF1ab and E genes of SARS-CoV-2 and performed a one-pot assay in which recombinase polymerase amplification (RPA) and Cas12a-based detection were combined in a single reaction. We noticed that several crRNAs exhibited faster fluorescence signal kinetics than others in the one-pot reaction ( Figure 7 ab). A detailed analysis of the top performing crRNAs revealed that all of them were designed to use the suboptimal PAMs (NTTV and TTNT) of Cas12a instead of the canonical PAM (TTTV) ( Figure 7 c). Based on these observations, we hypothesized that crRNAs with suboptimal PAMs speed up detection in one-pot assays, in which isothermal amplification of the target and Cas12a-mediated target cleavage occur simultaneously. To test this, substrates targeting spacers 4 and 5 of the ORF1ab gene were point mutated to convert their suboptimal PAMs to classical PAMs. As expected, crRNAs with suboptimal PAMs produced weaker and slower collateral activity than classical PAMs ( Figure 1 ab). We then used these substrates as RPA templates and performed both RPA and Cas12a-based assays in a one-pot assay. Compared to the spin-off activity assay, spacers 4 and 5 using suboptimal PAMs showed faster kinetics than canonical PAMs in a one-pot reaction ( Figure 1 cd).

[0114] To explore which types of suboptimal PAMs exhibit faster reactions in one-pot reactions, the substrates of spacers 4 and 5 of the Orf1ab gene, spacer 2 of the spike (S) gene of SARS-CoV-2, and spacer 1 of the HPV18 L1 gene were point mutated from TTTV to VTTV, TVTV, or TTVV. Comparison of the incidental activities and one-pot reactions of 120 suboptimal PAMs of the four spacers showed that more than 80% of the spacers with suboptimal PAMs exhibited faster reactions in one-pot reactions than those with classical PAMs, and most of the suboptimal PAMs that performed well were VTTV, TCTV, and TTVV ( Figure 1 eh, Figure 8-12 ). The protein structure of Cas12a shows that the PAM interaction domain mainly contacts the second nucleotide of the target strand; therefore, mutating the second nucleotide of the PAM from a pyrimidine to a purine may significantly weaken the activity of Cas12a ( Fig.13 ). In fact, some TATV and TGTV PAMs, but not TCTV PAMs, showed slower kinetics and reduced fluorescence signals in one-pot reactions; and consistently, these suboptimal PAMs all exhibited much lower incidental activities than the classical PAMs ( Figure 9-12 ). For spacers 4 and 5, TTTT PAM exhibited faster kinetics than TTTV PAM in a one-pot reaction, indicating that the fourth nucleotide of the PAM can also be modified to tune the activity of Cas12a ( Fig.14 ad). We then introduced two point mutations into the PAM. Introducing two PAM point mutations (TTTV to TTVT) into spacer 4 and part of spacer 5 resulted in faster kinetics in a one-pot reaction ( Fig.14 ad). We then examined other suboptimal PAMs with two point mutations. VVTV and VTVVPAMs with spacers 4 and 5 showed reduced kinetics and signal in one-pot reactions and incidental activity ( Fig.14 Interestingly, the mutation from TTTV to TCCV resulted in a superior reaction in a one-pot reaction ( Fig.14 h). This may be because the PAM of Cas12a can tolerate T to C mutations; TCCV has been characterized as a functionally suboptimal PAM (PMID: 28781234). CCCV with three point mutations is also considered a suboptimal PAM, and in fact, CCCA is faster than the classical PAM in a one-pot reaction ( Fig.14ij). As a negative control, another sequence AGCA with three point mutations of the PAM sequence showed minimal activity in the one-pot reaction. In conclusion, it suggests that subtle incidental activity levels are crucial in the one-pot reaction. Here, we conclude that most VTTV, TCTV, and TTVV, as well as some TRTV, TTNT, and YYYN (except TTTV) PAMs, outperform classical PAMs in the one-pot reaction.

[0115] To further demonstrate that the use of suboptimal PAM sequences can speed up one-pot reactions, we synthesized crRNAs targeting the E and S genes of SARS-CoV-2. All of these crRNAs reacted faster on substrates with suboptimal PAMs than on those with classical PAMs in one-pot reactions, suggesting that the use of suboptimal PAMs may be a universal strategy to speed up Cas12a-based one-pot assays ( Fig.15 ).

[0116] Previous studies have shown that despite the simplicity of one-pot CRISPR diagnostics, their sensitivity is lower than that of a two-step approach, in which target amplification and CRISPR detection are performed sequentially. Therefore, we investigated whether the application of a suboptimal PAM could improve the sensitivity of one-pot assays. The detection limit of spacer 4 using the classical PAM was 234 fM dsDNA in a one-pot reaction; in contrast, the detection limit of spacer 4 using the suboptimal PAM was 2.34 fM dsDNA ( Figure 2 a and b). To compare the stability of the assays using suboptimal and classical PAMs, we repeated the experiment ten times under the same conditions, with two biological replicates each time. Using substrate (2340 fM concentration of dsDNA) and incubation times sufficient for both suboptimal and classical PAMs, the fluorescence signal from the suboptimal PAM group was highly consistent across all replicates; in contrast, the signal from the classical PAM group varied more than 10-fold across all replicates ( Figure 2 c). We then compared the detection limits and stability of two other crRNAs using either the classical or suboptimal PAM. Both crRNAs using the suboptimal PAM showed approximately 10- to 100-fold increased sensitivity and very consistent signal generation compared to the crRNA using the classical PAM, demonstrating improved sensitivity and stability of the suboptimal PAM in one-pot reactions ( Figure 2 di).

[0117] To investigate the dose effect of Cas12a / crRNA ribonucleoprotein (RNP) in one-pot reactions, we tested RNP doses ranging from 5.5 nM to 132 nM using either suboptimal or classical PAM assays. Reactions using suboptimal PAMs showed stable kinetics and consistent results, with RNP doses ranging from 22-132 nM ( Fig.16 a), whereas reactions using classical PAMs exhibited dramatic fluctuations in kinetic curves and highly variable signals even with small changes in RNP dosage ( Fig.16 b). These data further illustrate that the use of suboptimal PAMs is key to reproducible results in Cas12a-mediated one-pot assays.

[0118] Next, we sought to understand the mechanism behind the robust performance of suboptimal PAM-mediated one-pot detection. In a one-pot reaction, CRISPR detection and isothermal amplification compete with each other, and the final detection signal depends on target amplification to generate sufficient substrate for CRISPR detection. crRNA using suboptimal PAMs may have slower initial kinetics of CRISPR detection and thus bias the reaction towards isothermal amplification. To verify this possibility, we monitored amplicon generation in one-pot reactions. For spacer 4, target amplicons were first observed two minutes after a one-pot reaction using a suboptimal PAM, while in the classical PAM group, eight to ten minutes were required to identify amplicons ( Figure 3 a). In addition, at each time point, the amount of amplicons generated by the one-pot reaction using the suboptimal PAM and PRA alone was much greater than the amount of amplicons generated by the one-pot reaction using the classical PAM ( Figure 3 Consistently, the generation of amplicons from spacer 5 and two additional spacers also showed faster kinetics and higher amplicon amounts in the test using the suboptimal PAM compared to the test using the classical PAM, indicating stronger interference with RPA amplification when using the classical PAM ( Figure 3 b, Fig.17 ab). To further compare the isothermal amplification capabilities under Cas12a monitoring, one-pot reactions were performed using mixed substrates consisting of 50% suboptimal and 50% classical PAMs. Amplicon sequencing analysis revealed that within the first minute of the reaction, amplicons from suboptimal PAM substrates accounted for approximately 90% or more of the amplified products, supporting the view that the use of suboptimal PAMs is critical for promoting RPA amplification under the pressure of competitive Cas12a cleavage ( Figure 3 cd).

[0119] Cas12a-mediated substrate binding and subsequent cis-cleavage may interfere with RPA amplification. The time course of cis-cleavage activity for a constant amount of DNA substrate showed that cleavage of the classical PAM substrate was completed within 30 seconds, while cleavage of the suboptimal PAM substrate required 10-20 minutes to complete ( Figure 3 ef). Cas12a can bind suboptimal PAM substrates with reduced affinity 35We infer that the delayed cleavage is due to the weak binding of Cas12a to DNA substrates with suboptimal PAMs. Consistently, electrophoretic mobility shift assay (EMSA) analysis of Cas12 binding affinity showed reduced binding to substrates with suboptimal PAMs compared to canonical PAMs of spacers 4 and 5 ( Figure 3 gh).

[0120] To further elucidate the mechanism, we evaluated 120 PAMs (including suboptimal and classical PAMs) from four different spacers (HPV18 L1 gene spacer 1, Orf1ab spacer 4, Orf1ab spacer 5, and S gene spacer 2) for cis-cleavage activity, collateral activity, and one-pot reaction ( Figure 9-12 and 18). We identified that K 切割 There is a clear correlation with the performance of one-pot reactions ( Figure 4 ab, Table 6). We defined the one-pot reaction factor of 30 min on the X-axis as the criterion for judging whether a PAM performs well in a one-pot reaction. All twelve classical PAMs of these four spacers and one suboptimal PAM of Orf1ab spacer 4 had a reaction factor of 1.2–3.5 min. -1 High K 切割 , they perform well in incidental activities but poorly in one-pot reactions ( Figure 4 ab, Table 6). With minimal cis-cleavage (K 切割 0-0.1min -1 ) had the worst performance in both incidental activity and one-pot reaction. In contrast, the -1 The middle K 切割 The suboptimal PAM outperformed the classical PAM in a one-pot reaction ( Figure 4 ab, Table 6). These results suggest that cis-cleavage efficiency is a key factor in determining the performance of one-pot reactions. Due to excessive substrate consumption caused by classical PAM-mediated cis-cleavage, amplicon accumulation is slow and unstable, resulting in delayed or absent side effects. Although suboptimal PAMs with minimal cis-cleavage allow accumulation of amplicons (substrates), they fail to perform sufficient side effects. In contrast, PAMs with intermediate K 切割 The suboptimal PAMs allow substrate accumulation early in the isothermal reaction while maintaining considerable incident activity. We ranked the best performing suboptimal PAMs based on the time to reach half-maximal fluorescence in one-pot reactions (Table 6). Among the top 5 best performing suboptimal PAMs per spacer (20 PAMs in total), there were 12 VTTVs and 5 TCTVs. Therefore, we suggest that VTTV can be selected as the first choice for suboptimal PAMs, while TCTV is a good candidate in one-pot reactions.

[0121] Table 6, shown below, is a summary of PAMs ranked by one-pot performance and cis-cleavage activity. The 120 PAMs were ranked by comparing performance in one-pot reactions, where "one-pot reaction" represents the time to reach half-maximal fluorescence (min)* based on the adjusted ratio of the platform signal for each PAM, K 切割 Represents cis-cleavage activity. Table 6. One-pot reaction performance

[0122] Together, these data suggest a model for how suboptimal PAMs function to promote isothermal amplification and, thus, robust and sensitive detection in a one-pot reaction ( Figure 4 b). Given that CRISPR detection and isothermal amplification compete with each other in a one-pot reaction, the reduced binding affinity of Cas12a for suboptimal PAM substrates promotes a shift in the equilibrium from cleavage to amplification and thus generates sufficient amplicons for detection; in contrast, the stronger binding affinity of classical PAMs allows cleavage to outperform amplification and results in delayed or absent amplicon production, which accounts for the observed detection delays and instabilities.

[0123] We have shown that using a suboptimal PAM is more optimal for one-pot reactions. These results were obtained using an RPA kit (Amp Future). To test whether this conclusion is valid using a different RPA kit, we performed experiments using an RPA kit from TwistDx. The two RPA kits showed no difference in amplification sensitivity ( Fig.19 a). However, when we combined RPA and Cas12a in one reaction, the TwistDx kit showed much lower sensitivity and reduced fluorescence signal ( Fig.19 b). A small amount of Cas12a RNP (2 μL) was added to RPA (18 μL) for a one-pot reaction. This indicates that Cas12a is not fully compatible with the buffer environment that works well with TwistDx RPA. A recent study showed that LwaCas13a is also not fully compatible with the TwistDx RPA buffer environment. Arizti-Sanz et al. optimized the buffer to make it suitable for both RPA amplification and Cas13a activity, a method called SHINE. Inspired by this study, we increased the RNP dose in the reaction from 33.3nM to 100nM, 200nM, and 333nM. We found that a 3-6-fold increase in RNP can significantly improve the fluorescence curve of the one-pot reaction ( Fig.19c). Using this improved condition and the TwistDx kit, we compared the performance of the classic PAMs and suboptimal PAMs for four spacers. Similar to the Ample Future kit, the suboptimal PAMs using the TwistDx kit performed much better than the classic PAMs ( Fig.19 dg).

[0124] Therefore, we developed a flexible, accelerated, suboptimal PAM-based assay with enhanced sensitivity and reproducibility (FASTER). To determine whether FASTER could detect DNA viruses, we tested the method with the double-stranded DNA virus human cytomegalovirus (HCMV). We first compared the sensitivity of FASTER with qPCR using a plasmid containing the UL55 gene sequence of HCMV as a substrate. Both assays showed the same limit of detection, i.e., 5.953 × 10 -4 amol plasmid / reaction (equal to 29.765aM concentration in qPCR and 19.843aM in FASTER) ( Figure 5 ab). Strikingly, for all tested concentrations, the fluorescence signal of FASTER began to appear at approximately 6-10 minutes and reached half-maximum at approximately 9-15 minutes ( Figure 5 b). Notably, this rate is at least 2 to 3 times faster than all published CRISPR-mediated one-pot assays using target amplification. We then used FASTER to measure HCMV viral samples. The results showed a limit of detection of 24 copies / reaction, which is comparable to the detection limit of qPCR ( Figure 5 cd). To enable broader application of FASTER, we used simple UV light instead of fluorescence spectroscopy to measure the signal. At the 10-minute time point, all but the lowest virus concentration showed a positive signal for the UV assay, while at 15 minutes, the sample with the lowest viral copy number (equal to a qPCR Ct value of 36) was clearly positive ( Figure 5 We also combined FASTER with lateral flow assay strips, and this combination was able to detect virus samples with Ct values ​​of 33-34 ( Figure 5 g). These results are consistent with previous studies showing that lateral flow strip assays are less sensitive than their corresponding fluorescence signal-based assays. Lateral flow readouts require opening the tubes to add buffer and strips, an additional step that increases hands-on and waiting time as well as the risk of cross-contamination. Since the fluorescence readout stimulated by simple UV light is simpler, faster, and more sensitive, we decided to use it for FASTER to detect SARS-CoV-2 samples.

[0125] One advantage of FASTER is that it greatly expands the available choices for crRNAs, since there are more suboptimal PAMs than canonical PAMs. Spacers using VTTV, TCTV, and TTVV PAMs are likely to perform well in a one-pot reaction, making the number of available suboptimal PAMs theoretically 7-fold higher than the number of canonical PAMs (21 combinations versus 3 combinations) ( Figure 6 a, Fig. 20 a). In addition, some other suboptimal PAMs, such as TRTV, TTNT, and YYYN (except TTTV), may also work better than the classical PAMs, making the choice of spacer even more flexible ( Fig. 20 bc). Relaxed criteria for PAM selection are particularly important for developing assays for virus detection. Although there are more than 1000 classical PAMs for Cas12a in SARS-CoV-2, only a limited number of classical PAMs can be used for virus detection assays given the following selection criteria: 1) in conserved regions; 2) in high-copy genes; 3) active crRNA; 4) compatible with robust primers for isothermal amplification. Therefore, the expanded selection of suboptimal PAMs makes FASTER more flexible in assay optimization and application to new virus strains.

[0126] Finally, we applied FASTER to detect SARS-CoV-2. We first compared the sensitivity of FASTER based on RPA / Cas12a / suboptimal PAM and STOPCovid based on LAMP / Cas12b using a DNA fragment encoding the N gene of SARS-CoV-2. FASTER was approximately 100 times more sensitive than STOPCovid, and FASTER was 3 times faster than STOPCovid in detecting DNA samples (13 minutes vs. 40 minutes, time to half-maximum fluorescence) ( Figure 6 be). We then examined the ability of FASTER to detect RNA samples by combining RT-RPA and Cas12a. Our initial data showed that FASTER was not as sensitive as RT-qPCR in detecting RNA samples. We speculated that the RT step was the rate-limiting step, even though RNase H was added to the reaction. To improve the efficiency of the RT step, we first increased the reaction temperature from 37°C to 42°C, because RT enzymes generally perform better at higher temperatures, and both RPA and Cas12a are activated at 42°C. In fact, FASTER performed well at 42°C ( Fig.21 a). Similar to RT-qPCR, RT-RPA usually uses its reverse primer as the RT primer. We hypothesized that RPA reverse primers longer than 30 nt may be ineffective for the RT step. In fact, the RT efficiency of RPA reverse primers is 6 times lower than that of qPCR reverse primers ( Fig.21b). Therefore, we added an additional 18nt primer as RT primer and reduced the concentration of RPA reverse primer to prevent it from interfering with the RT process. The combination of reaction at 42°C, addition of additional short RT primer and reduction of RPA reverse primer concentration significantly improved RT efficiency and overall FASTER performance for RNA samples ( Fig.21 ad). The ability of FASTER and STOPCovid to detect RNA was compared using in vitro transcribed RNA fragments of the SARS-CoV-2N gene. FASTER was about 100 times more sensitive than STOPCovid in detecting RNA and about 2.5 times faster ( Figure 6 be).

[0127] We directly compared the limits of detection (LOD) of FASTER and RT-qPCR, which is considered the current gold standard by the Centers for Disease Control and Prevention (CDC). We first determined the LOD of RT-qPCR using a commercially available SARS-CoV-2 pseudovirus as a standard. The LOD of RT-qPCR was 1 cp / μL ( Fig. 22 ab), which is consistent with the results published by the CDC. We then compared the LODs of FASTER and RT-qPCR and identified that the LOD of FASTER was 1 cp / μL ( Fig. 22 c), which is comparable to the LOD of RT-qPCR.

[0128] We evaluated the performance of FASTER in SARS-CoV-2 positive and negative clinical samples and compared its performance with that of STOPCovid. A total of 104 SARS-CoV-2 positive nasopharyngeal swab samples (48 unextracted samples and 56 extracted samples) and 100 SARS-CoV-2 negative samples were used, and the Ct values ​​of these positive nasopharyngeal swab samples ranged widely (from 18.1 to 35.8). FASTER had a sensitivity of 94.2% and a specificity of 100.0%, and it was able to detect samples with a Ct value of 35.8 (according to Fig. 22 The standard curve of RT-qPCR in b is 1.2 cp / μL) Figure 6 fh). Positive signals appeared as early as 10 minutes, and all positive samples showed signals at 15 minutes ( Figure 6 h, Fig.23 a). The signal can be detected by UV light or simple blue light devices ( Fig.23 b). In contrast, STOPCovid.v1 was unable to stably detect samples with Ct values ​​higher than 31.0, resulting in a sensitivity of 78.8% ( Fig.24ac). Finally, to evaluate the specificity of FASTER, we tested several common human coronaviruses, including MERS, HKU1, 229E, NL63, and OC43, by RPA amplification, incidental activity testing, and one-pot reactions. These results showed no cross-reactivity with other common viruses ( Fig.25 ad).

[0129] One-pot detection using suboptimal PAMs can be applied to other members of the Cas12a family and other class II type V effectors. It will be interesting to explore whether other Cas proteins can show better speed than Cas12a using suboptimal PAMs in one-pot reactions. Therefore, we also provide a rapid and sensitive detection method using Cas protein mutants. We mutated the amino acids that form hydrogen bonds with PAM on the LbCas12a protein to alanine, and then used the mutant protein to target the classical PAM to establish a rapid one-step detection. As we expected, the two mutants, K595A and K595A&Y542A, reached the plateau faster than the wild-type protein in the one-step reaction, especially K595A, which could reach the peak within 20 minutes ( Fig. 27 ad). In addition, the sensitivity of the mutant was also improved by 100 times compared with the wild type. The detection limit of the K595A mutant was 16.457aM N gene dsDNA, while the wild type could only identify 1645.7aM dsDNA ( Fig. 27 eg).

[0130] The one-step method of Cas12a and RPA reacts at 37°C-42°C. A reverse transcription step is included for detecting RNA virus samples, and higher temperatures may be beneficial for this step. High temperature resistant methods such as Cas12b can be combined with high temperature isothermal amplification methods such as LAMP. We mutated the PAM associated with residues 478G, 396K, and 403Q to alanine in Cas12b, and the mutant is abbreviated here as 3M (a total of 3 point mutations). The results of cis-cleavage and trans-cleavage showed that the activity of 3M was indeed weaker than that of WT ( Fig.28 ab). In the RPA-mediated one-step method, the reaction rate of 3M was significantly faster than that of WT ( Fig.28 c). Next, we tested it in a LAMP-mediated one-pot reaction, and the results were consistent with the RPA-mediated one-pot reaction. Not only was the reaction speed faster, but the sensitivity was also improved by 10 times ( Fig.28 In short, using Cas12b 3M protein, 164.57aM sample can be clearly detected within 25 minutes ( Fig.28 f).

[0131] In conclusion, the FASTER assay developed in our study showed a shorter incubation time than STOP and was more sensitive than Cas13a-based assays without preamplification. The FASTER assay is the first CRISPR-mediated assay that combines the following features: speed, sensitivity, reliability, and flexibility. ***

[0132] The scope of the present disclosure is not limited by the specific embodiments, which are intended only to illustrate various aspects of the present disclosure, and any functionally identical compositions or methods are within the scope of the present disclosure. It should be clear to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, it is contemplated that the present disclosure covers modifications and variations of the present disclosure, provided that they are within the scope of the appended claims and their equivalents.

[0133] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A use of a mixture in the preparation of a diagnostic reagent for one-step detection of a target polynucleotide, the mixture comprising (a) a polymerase, (b) deoxynucleoside triphosphates (dNTPs), (c) a primer for amplifying the target polynucleotide, (c) a CRISPR-associated (Cas) nuclease, and (d) a guide RNA comprising a spacer segment complementary to a target segment on the target polynucleotide, wherein the mixture enables the polymerase to effectively amplify the target polynucleotide, while the Cas nuclease is capable of cleaving the amplified target polynucleotide, wherein the target segment of the guide RNA includes or is adjacent to a protospacer adjacent motif (PAM) sequence that can be recognized by the Cas nuclease, and the PAM sequence is non-canonical and reduces interaction with the Cas nuclease and / or reduces the cleavage activity of the Cas nuclease.

2. The use of method 1 as claimed in claim 1, wherein the Cas nuclease is LbCas12a, and the PAM sequence is selected from the group consisting of NTTV, TNTV, TTNV, TTNT, VTTT, TVTT, VVTT, VTVT, VNVV, NVNV, NVVV, VNTV, NTVV, TNVV, YYYN and VVNV, wherein N represents any nucleotide and V represents a non-T nucleotide.

3. The use of claim 1, wherein the Cas nuclease is AapCas12b, and the PAM sequence is selected from the group consisting of: VTN, TTN, TVN, NVN and VVN, wherein N represents any nucleotide and V represents a non-T nucleotide.

4. The use according to any one of claims 1 to 3, wherein at least one dNTP is modified.

5. The use according to claim 4, wherein the modification is performed with a group selected from the group consisting of phosphoryl, biotin, digoxigenin, amino, thiol, phosphorothioate and methyl.

6. The use of claim 4, wherein at least one dNTP is replaced by the following or at least one nucleotide in the primer is as follows: deoxyuridine triphosphate, deoxyinosine triphosphate, pseudouridine triphosphate, methylpseudouridine triphosphate, 2-aminopurine, 5-bromo-dU or ribonucleoside triphosphate (rNTP).

7. The use according to claim 6, wherein the rNTP is modified.

8. The use according to claim 6 or 7, wherein the modification is performed with a group selected from the group consisting of phosphoryl, biotin, digoxigenin, amino, thiol, phosphorothioate and methyl.

9. The use according to any one of claims 1 to 8, wherein the guide RNA comprises a truncated or 5' / 3' DNA- / RNA-extended CRISPR RNA (crRNA) including the spacer fragment.

10. The use according to claim 9, wherein the spacer fragment is 19 nucleotides or shorter, preferably 18, 17, 16, 15, 14, 13, 12, 11, 10 nucleotides or shorter.

11. The use of claim 9 or 10, wherein the truncated or extended crRNA comprises a truncated or extended hairpin structure sequence.

12. The use according to any one of claims 1 to 11, wherein the guide RNA comprises a truncated trans-activating CRISPR RNA (tracrRNA) sequence.

13. The use according to any one of claims 1 to 12, wherein the guide RNA comprises a region complementary to at least a portion of the spacer fragment or the hairpin.

14. The use according to any one of claims 1 to 13, wherein at least one nucleotide in the spacer is not a standard ribonucleotide, which is preferably located at nucleotides 1 to 25 in the spacer.

15. The use of claim 14, wherein at least one nucleotide in the spacer fragment is selected from the group consisting of: deoxynucleotides, locked nucleic acids (LNA), bridged nucleic acids (BNA), deoxyuridine, deoxyinosine, pseudouridine, methylpseudouridine and modified nucleotides, wherein the modification is carried out with a group selected from the group consisting of: phosphoryl, biotin, digoxigenin, amino, thiol, phosphorothioate, methyl, 2'-O-methyl-3'-phosphonoacetate (MP), 2'O-methoxyethyl (MOE), fluoro (F), S-constrained ethyl, 2'-O-methyl-PS (MS) and 2'-O-methyl-thio PACE (MSP).

16. The use of any one of claims 1 to 15, wherein the spacer fragment comprises 1, 2, 3, 4, 5 or 6 internal mismatches with the target fragment.

17. The use according to any one of claims 1 to 16, wherein the Cas nuclease is selected from the group consisting of Cas12a, Cas12b, Cas12c, Cas12d, Cas12e (CasX), Cas12f, Cas12k, Cas13a, Cas13b, Cas13c, Cas13d, Csm6, Csm3, Cas14a, Cas14b and Cas14c.

18. The use of claim 17, wherein the Cas nuclease is selected from the group consisting of: AsCas12a, FnCas12a, MbCas12a, Lb3Cas12a, Lb2Cas12a, BpCas12a, PeCas12a, PbCas12a, SsCas12a, CMtCas12a, EeCas12a, LiCas12a, PcCas12a, PdCas12a, PmCas12a, ArCas12a, HkCas12a, ErCas12a, BsCas12a, LpCas12a, PrCas12a 2a, PxCas12a, AapCas12b, AmCas12b, AacCas12b, BsCas12b, BvCas12b, BthCas12b, BhCas12b, AkCas12b, EbCas12b, LsCas12b, Mi1Cas 12f2, Mi2Cas12f2, Un1Cas12f1, Un2Cas12f1, AuCas12f2, PtCas12f1, AsCas12f1, RuCas12f1, SpCas12f1, CnCas12f1 (Cas14a) of Cas12f; ShCas12k(CAST), AcCas12k, LwaCas13a, LbaCas13a, LshCas13a, PprCas13a, EreCas13a, LneCa3a, CamCas13a, RcaCas13a, HheCas13a, LbuCas13a, LseCas13a, LbmCas13 a, LbnCas13a, RcsCas13a, RcrCas13a, RcdCas13a, CgCas13a, Cg2Cas13a, LweCas13a, LbfCas13a, Lba4Cas13a, Lba9Cas13a, LneCas13a, HheCas13a, RcaCas13a of Cas13a; BzCas13b, PbCas13b, PspCas13b, RanCas13b, PguCas13b, PsmCas13b, CcaCas13b, AspCas13b, PauCas1 3b, Pin2Cas13b, Pin3Cas13b, RspCas13d, RfxCas13d, EsCas13d, AdmCas13d, TtCsm6, EiCsm6 and LsCsm6.

19. The use according to any one of claims 1 to 18, wherein the Cas nuclease is sequence engineered.

20. The use of claim 19, wherein the sequence engineering changes the activity of the Cas nuclease in forming ribonucleoprotein (RNP) or binding substrate nucleic acid.

21. The use of claim 20, wherein the sequence-engineered Cas nuclease is LbCas12a having one or more amino acid deletions or substitutions at residues selected from the group consisting of: Lys15, Thr16, Arg18, Lys20, Lys51, Asn157, Arg158, Arg174, Lys253, Gln264, Lys278, Leu281, Arg386, Lys390, Lys464, Arg508, Lys520, Lys707, Ser710, Thr713, His714, Gly715, Thr716, Asn718, His720, Arg747, Ala766, Asn767, Lys768, Asn769, Asn772, Lys774, Thr777, Tyr781, Asp786, Arg788, Gln793, Asn808, Tyr872, Glu898, Lys953 and Lys960.

22. The use of claim 20, wherein the sequence-engineered Cas nuclease is AsCas12a having one or more amino acid deletions or substitutions at residues selected from the group consisting of: Lys414, Gln286, Lys273, Lys369, Gly270, His479, Asn515, His479, Arg518, Gln956, Trp382, Arg192, Lys307, Leu310, Arg176, Lys51, Asn175, Tyr47, Glu786, His s872, Lys530, His761, Thr16, Lys15, Lys1022, His977, Lys1029, Lys757, His856, Leu807, Met806, Asn808, Lys852, Ile858, Lys810, Lys809, Arg863, Lys943, Tyr940, Asp966, Arg790, Lys748, Arg18, Lys752, Ser973, Leu760, Arg313, Arg955, and Asn759.

23. The use of claim 20, wherein the sequence-engineered Cas nuclease is AapCas12b having one or more amino acid deletions or substitutions at residues selected from the group consisting of: Lys4, Ser5, Lys7, Lys9, Trp391, Arg415, Ser442, Met443, Gln446, Arg484, Arg485, Tyr501, Asn503, Phe600, His614, Gln618, Arg731, Arg734, Val737, Arg738, Arg74 2. Pro743, Lys744, Ile745, Arg746, Arg746, Val753, Gly754, Gly755, Leu764, Asn766, Gln767, Arg792, Ala794, Thr796, His800, His803, Asp807, Lys810, Lys811, Asp814, Arg815, Glu819, Tyr825, Trp835, Tyr839, Asn881, Gln882, Gln973, Leu978 and Gln982.

24. The use of claim 20, wherein the sequence-engineered Cas nuclease is AacCas12b having one or more amino acid deletions or substitutions at residues selected from the group consisting of Leu978, Gln982, Gln973, His514, Phe600, Tyr839, Arg815, Glu819, Gln618, Arg415, Met443, Ala794, Arg792, Arg738, Arg731, Arg734, Thr796, Arg746, Leu764, Arg738, Gln767, Val737, His800, Pro743, Val753, Gly754, Asn756, Asp807, His803, Arg484, Tyr501, Lys9, Ser442, Arg742, Lys744, Ile745, Gly755, Arg746, Gln446, Lys810, Ser5, Asn503, Asn881, Lys7, Trp391, Arg485, Tyr825, Lys811, Trp835, Gln882, Asp814 and Lys4.

25. The use of claim 20, wherein the sequence-engineered Cas nuclease is LbuCas13a having one or more amino acid deletions or substitutions at residues selected from the group consisting of: Met1, Lys2, Val3, Thr4, Lys5, Ser10, His11, His11, Asn139, Lys140, Asn142, Ser143, Ser147, Asn151, Arg172, Tyr176, Arg224, His228, Arg233, Lys237, Tyr245, Gln271, Tyr274, Lys275, Tyr276, His294, Glu297, Ser301, Lys305, Tyr 307, Arg311, Lys319, Arg322, Lys336, Asn339, Lys340, Ser363, Ala367, Glu371, Phe375, Asn378, Asn547, Arg547, Asn549, Ser555, Lys558, Tyr601, His771, Ser780, Lys783, Ala787, Asn804, Asn804, Arg809, Arg857, His901, Tyr938, Lys942, His962, Arg963, Arg973, Arg1072, Asn1083, Lys1087, Ser1088, Phe1102, and Ala1106.

26. The use of claim 19, wherein the sequence engineering changes the conformation of the Cas nuclease.

27. Application as claimed in claim 25, wherein the sequence-engineered Cas nuclease is LbCas12a, which has one or more amino acid deletions or substitutions at a residue selected from the group consisting of: Lys457, Val511, Thr512, Gln888 and Try890, or is LbuCas13a, which has one or more amino acid deletions or substitutions at a residue selected from the group consisting of: Lys2, Lys5, Gln371, Phe375, Lys783 and His962.

28. The use of claim 19, wherein the sequence engineering changes the activity of the Cas nuclease in interacting with a PAM sequence.

29. The use of claim 28, wherein the sequence-engineered Cas nuclease is LbCas12a having one or more amino acid deletions or substitutions at residues selected from the group consisting of: Lys121, Thr148, Thr149, Trp534, Asp535, Lys538, Tyr542, Lys595, Ser599, Lys600, Lys601, Try616, Try646, Trp649 and Gly740, or AapCas12b having one or more amino acid deletions or substitutions at residues selected from the group consisting of: One or more amino acid deletions or substitutions are present at: Lys209, Lys141, Ala142, Lys145, Asn144, Gly143, Asn400, Lys396, Gln118, Gly478, Arg507, Gln403, Arg208, Gln211, Ala212, Val213, Arg218, Val134, Gly135, Leu137, Gly136, Gln119, Arg122, Gly143, Asn144 and Arg150, Arg147.

30. The use of claim 28, wherein the sequence-engineered Cas nuclease is AsCas12a having one or more amino acid deletions or substitutions at residues selected from the group consisting of: Lys164, Thr166, Thr167, Ala135, Lys607, Lys613, Asn631, Tyr687, Asn547, Lys689, Asp545, Lys548, Lys780, Asn782, and Gly783.

31. Use as claimed in claim 28, wherein the sequence-engineered Cas nuclease is AacCas12b, which has one or more amino acid deletions or substitutions at residues selected from the group consisting of: Lys209, Lys141, Ala142, Lys145, Asn144, Gly143, Asn400, Lys396, Gln118, Gly478, Arg507, Gln403, Arg208, Gln211, Ala212, Val213, Arg218, Val134, Gly135, Leu137, Gly136, Gln119, Arg122, Gly143, Asn144 and Arg150, Arg147.

32. The use of claim 19, wherein the sequence engineering is located at a conserved residue within the REC1 domain, REC2 domain, Helix I domain or Helix II domain.

33. Use as claimed in claim 19, wherein the sequence engineering is located at a residue within the REC lobe or RuvC domain of the Cas nuclease, which residue is positively charged or negatively charged, or is capable of forming a hydrogen bond with the target polynucleotide.

34. The use of claim 33, wherein the residue is one or more of: Asn160, Lys167, Ser168, Asn256, Asn260, Ser286, Ser288, Trp355, Lys514, Ile896, Lys897, Gln944, Lys945, Phe983, Gly740 or Lys984 of LbCas12a; Ser376, Asn282, Asn278, Arg301, Thr315, Lys524, Arg955, Arg951, Ile964, Lys965, Gln1014, Phe1052, Ala1053, Ser186, Asn178, Lys603, Gln784, Lys780, Gly783, Ser1051, Gln1013, Lys968, Gly263, or Trp382 of AsCas12a; Lys1066, Lys895, Lys1281, Lys1287, Lys1069, Arg1016, Phe1010, Arg1014, Asn1288, Leu329, Asp331, Ser330, Gln327, Lys326, Leu324, Leu1008, Gln1006, Asp917, Asn1009, Arg1014, Lys1021, Arg1016, Lys1013, Gln309, Asn305, Leu306, Glu302 or Asn301 of FnCas12a, Arg332, Arg900, Phe897, Ser898, Ser899, Asp570, Glu848, Asp977, Arg911, Leu573, Arg574, Arg913, Asn110 of AapCas12b 1. Gln1093, Arg859, Tyr853, Gln866, Arg331, Gly955, Trp930, Ser5, Pro86, Gln109, Ala117, Lys141, Ala142, Lys145, Ar g208, Lys209, Gln211, Ala212, Val213, Arg218, Gln222, Ser233, Trp234, Arg237, Leu281, Lys284, Glu285, His289, Thr292, Arg294, Arg297, Ser335, Gln403, Ser505, Arg785, Lys789, Phe793, Arg798, Lys805, Phe855, Ser862, Arg873, or Gly874; Ser5, Pro86, Gln109, Ala117, Lys141, Ala142, Lys145, Arg208, Lys209, Gln211, Ala212, Val213, Arg218, Gln222, Ser233, Trp234, Arg237, Leu281, Lys284, Glu285, His289, Thr292, Arg294, Arg297, Ser335, Gln403, Ser505, Arg78 of AacCas12b 5. Lys789, Phe793, Arg798, Lys805, Phe855, Ser862, Arg873, Gly874, Arg332, Arg900, Phe897, Ser898, Ser899, Asp570, Glu848, Asp977, Arg911, Leu573, Arg574, Arg913, Asn1101, Gln1093, Arg859, Tyr853, Gln866, Arg331, Gly955, or Trp930; or Lys2, Arg41, Lys47, Lys86, His473, His477, Gln519, Ser522, Thr557, Asp590, Lys597, Gln659, Arg809, Val810, Lys855, Arg857, Gln904, Glu996, Phe995, Asn997, Lys998, Gln1007 or Arg1135 of LbuCas13a.

35. Use as claimed in claim 19, wherein the sequence engineering is located at a conserved residue in the HNH motif, REC lobe or Nuc lobe of the Cas nuclease.

36. Use according to any one of claims 19 to 35, wherein the sequence is engineered to be a non-conservative substitution or to be substituted with alanine.

37. The use of any one of claims 1 to 36, wherein the mixture further comprises a polymerase activator that increases the amplification kinetics of the polymerase.

38. The use according to any one of claims 1 to 37, wherein the mixture further comprises dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), tween 20, a proteinase K inhibitor or a nuclease inhibitor.

39. The use according to any one of claims 1 to 38, wherein the mixture further comprises an agent selected from Table F.

40. The use according to any one of claims 1 to 39, wherein the target polynucleotide is single-stranded RNA, double-stranded RNA, single-stranded DNA or double-stranded DNA.

41. The use according to claim 40, wherein the target polynucleotide is viral DNA or RNA, or genomic DNA containing SNP (single nucleotide polymorphism).

42. The use of claim 40, further comprising a detectable tag that is activated when the target polynucleotide is cleaved by the Cas nuclease.

43. A kit or package for detecting a target polynucleotide, the kit or package comprising (a) a polymerase, (b) deoxynucleoside triphosphates (dNTPs), (c) primers for amplifying the target polynucleotide, (d) a CRISPR-associated (Cas) nuclease, and (d) a guide RNA comprising a spacer complementary to a target fragment on the target polynucleotide, wherein the polymerase can effectively amplify the target polynucleotide and the Cas nuclease can cleave the amplified target polynucleotide.

44. A kit or package for detecting a target polynucleotide, the kit or package comprising (a) a polymerase, (b) deoxynucleoside triphosphates (dNTPs), and (c) primers for amplifying the target polynucleotide, wherein at least one primer comprises a suboptimal PAM sequence for a Cas nuclease, or wherein at least the dNTP or primer is modified to reduce cleavage or binding by the Cas nuclease.

45. A kit or package for cleaving a target polynucleotide, the kit or package comprising (a) a CRISPR-associated (Cas) nuclease, and (b) a guide RNA comprising a spacer segment complementary to a target segment on the target polynucleotide, wherein the guide RNA has reduced binding to the target polynucleotide or reduced cleavage of the target polynucleotide compared to a standard guide RNA.

46. ​​A mutant Cas nuclease having (a) reduced activity in forming ribonucleoprotein (RNP), (b) an altered conformation, (c) reduced activity in interacting with a target PAM sequence, or (d) reduced binding to a target polynucleotide to be cleaved.

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