CN111778230A

CN111778230A - Buffer system suitable for Cas12 protein and application thereof

Info

Publication number: CN111778230A
Application number: CN202010694640.2A
Authority: CN
Inventors: 段志强; 陈莹
Original assignee: Shandong Shunfeng Biotechnology Co Ltd
Current assignee: Shandong Shunfeng Biotechnology Co Ltd
Priority date: 2020-07-17
Filing date: 2020-07-17
Publication date: 2020-10-16
Also published as: CN113416719A; CN113416719B

Abstract

The invention provides a buffer system suitable for a Cas12 protein and application thereof, and particularly relates to a buffer system suitable for a Cas12 protein, wherein the buffer system comprises a pH buffer solution; a divalent cation; reducing agents and stabilizers. Wherein the pH buffer is a pH buffer that maintains the pH at 7.0 to 9.0; the divalent cations are magnesium ions; the reducing agent is selected from one or any of DTT, beta-mercaptoethanol and TCEP; the stabilizer is selected from one or more of BSA, glycerol and PEG.

Description

Buffer system suitable for Cas12 protein and application thereof

Technical Field

The invention relates to a buffer system, in particular to a buffer system suitable for Cas12 protein and application thereof.

Background

The CRISPR/Cas system is known as a Clustered Regularly Interspaced Short Palindromic Repeats and associated protein system (Clustered regulated interleaved Short Palindromic Repeats/CRISPR-associated proteins). Is an acquired immune system in a bacterial body and is used for resisting exogenous DNA, plasmids, phages and the like invading bacteria. Based on this mechanism, scientists developed CRISPR/Cas technology to use RNA-guided Cas nucleases to perform modification editing of specific sites of various species genomes. The currently discovered CRISPR/Cas systems can be broadly divided into two categories, Class1 (including type I, III, and IV), and Class 2 (including type II, type V, and type VI). Wherein the Cas12 protein in TypeV can specifically recognize target single-stranded/double-stranded DNA sequence under the guidance of the artificially designed gRNA, so that trans activity is activated, and ssDNA/ssRNA (reporter, nucleic acid detector) in a non-differential cleavage reaction system is activated.

The applicant finds that buffer systems with different proportions have great influence on the activity of the Cas protein when studying the in vitro activity of the Cas protein, and provides an optimized buffer system in order to improve the application efficiency of the Cas protein.

Disclosure of Invention

In one aspect, the invention provides a buffer system suitable for a Cas12 protein, the buffer system comprising a pH buffer; a divalent cation; a reducing agent; and a stabilizer.

In one embodiment, the pH buffer is a pH buffer that maintains the pH of the solution at 7.0-9.0.

In a preferred embodiment, the pH buffer is a pH buffer that maintains the pH of the solution at 7.6-8.5.

In a most preferred embodiment, the pH buffer is a pH buffer that maintains the solution pH at 7.9.

In one embodiment, the pH buffer is selected from one or any of the following buffers: tris buffer, ACES buffer, PIPES buffer, PBS buffer, MOPAS buffer, MOPSO buffer, Bis-Tris Propane buffer, BES buffer, MOPS buffer, TES buffer, HEPES buffer, DIPSO buffer, MOBS buffer, TAPSO buffer, Trizma buffer, HEPSO buffer, POPSO buffer, TEA buffer, EPPS buffer, Tricine buffer, Gly-Gly buffer, Bicine buffer, HEPES buffer, TAPS buffer, AMPD buffer, TABS buffer, AMPSO buffer, CHES buffer.

In a preferred embodiment, the pH buffer is Tris buffer or HEPES buffer.

Preferably, the Tris buffer is selected from a Tris-hydrochloric acid buffer and a Tris-acetic acid buffer, and preferably, the Tris-acetic acid buffer.

In one embodiment, the divalent cation is a divalent magnesium ion.

In one embodiment, the divalent magnesium ion is provided by a salt containing the magnesium ion, the salt being selected from magnesium chloride, magnesium acetate, magnesium sulfate or magnesium citrate, preferably, magnesium acetate.

In one embodiment, the stabilizer is selected from one or any several of BSA, glycerol and PEG;

in one embodiment, the reducing agent is selected from one or any of DTT, beta-mercaptoethanol, TCEP hydrochloride;

in one embodiment, the final concentration of the pH buffer is 5-100mM, preferably, 10-50mM, more preferably, 40 mM.

In one embodiment, the divalent cation has a final concentration of 5-400mM, preferably, 10-200mM, more preferably, 30-150mM, more preferably, 30 mM.

In one embodiment, the final concentration of the stabilizing agent is 5-400ug/mL, preferably 10-200ug/mL, more preferably 120 ug/mL.

In one embodiment, the reducing agent is present at a final concentration of 1-100mM, preferably 5-50mM, more preferably 5-20mM, more preferably 12 mM.

In one embodiment, the solvent of the buffer system is an organic solvent or an inorganic solvent, preferably the solvent is an inorganic solvent, more preferably the solvent of the buffer system is water.

On the other hand, the invention also provides application of the buffer system in improving in vitro activity of the Cas12 protein.

In another aspect, the invention also provides a method of increasing in vitro activity of a Cas12 protein, the method comprising providing the above-described buffer system suitable for a Cas12 protein, and placing the Cas12 protein in the buffer system to exhibit in vitro activity.

Further, the in vitro activity includes cis cleavage activity and trans cleavage activity.

On the other hand, the invention also provides application of the buffer system in improving the nucleic acid detection efficiency of the Cas12 protein.

The nucleic acid detection is achieved by the trans cleavage activity of the Cas12 protein.

In another aspect, the invention also provides a method for detecting nucleic acid by using the Cas12 protein, which comprises providing the above buffer system suitable for the Cas12 protein, and placing the Cas12 protein in the buffer system to detect nucleic acid.

The above nucleic acid detection is achieved by trans cleavage activity of Cas12 protein.

In one embodiment, the Cas12 protein cleaves a single-stranded nucleic acid detector by trans cleavage activity, which can exhibit a detectable signal after cleavage, thereby achieving the purpose of nucleic acid detection, as described in chinese patent application CN 2020104781299; the detectable signal is achieved by any one of: vision-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase transition/dispersion, electrochemical detection, and semiconductor-based detection.

The buffer system provided by the invention can improve the efficiency of the single-stranded nucleic acid detector for the cleavage of the Cas12 protein, and can display a detectable signal in 3 minutes or less at the fastest speed.

In the present invention, the single-stranded nucleic acid detector includes a single-stranded DNA, a single-stranded RNA, or a single-stranded DNA-RNA hybrid. In other embodiments, the single-stranded nucleic acid detector comprises a mixture of any two or three of single-stranded DNA, single-stranded RNA, or single-stranded DNA-RNA hybrids, e.g., a combination of single-stranded DNA and single-stranded RNA, a combination of single-stranded DNA and single-stranded DNA-RNA hybrids, and a combination of single-stranded RNA and single-stranded DNA-RNA.

In a preferred embodiment, the single stranded nucleic acid detector is a single stranded oligonucleotide detector.

The single-stranded nucleic acid detector does not hybridize to the gRNA.

In other embodiments, the single-stranded nucleic acid detector comprises one or more modifications, such as base modifications, backbone modifications, sugar modifications, and the like, to provide new or enhanced features (e.g., improved stability) to the nucleic acid. Examples of suitable modifications include modified nucleic acid backbones and non-natural internucleoside linkages, and nucleic acids having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified oligonucleotide backbones containing phosphorus atoms therein include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates. In some embodiments, the single stranded nucleic acid detector comprises one or more phosphorothioate and/or heteroatomic nucleotide linkages. In other embodiments, the single stranded nucleic acid detector can be a nucleic acid mimetic; in certain embodiments, the nucleic acid mimetics are Peptide Nucleic Acids (PNAs), another class of nucleic acid mimetics is based on linked morpholino units having a heterocyclic base attached to a morpholino ring (morpholino nucleic acids), and other nucleic acid mimetics further include cyclohexenyl nucleic acids (CENAs), further including ribose or deoxyribose chains.

In another aspect, the invention also provides a method of using a Cas12 protein to bind and/or cleave nucleic acid in vitro, the method comprising providing the above-described buffer system suitable for a Cas12 protein, placing the Cas12 protein in the buffer system to bind and/or cleave nucleic acid in vitro. The nucleic acid includes single-stranded nucleic acid and double-stranded nucleic acid.

Under the action of gRNA, Cas12 protein can bind to target nucleic acid, can cleave target nucleic acid by cis cleavage activity, and can cleave arbitrary single-stranded nucleic acid by trans cleavage activity.

In another aspect, the invention also provides a nucleic acid detection kit comprising a Cas12 protein and the above-described buffer system suitable for a Cas12 protein.

Further, the kit also comprises gRNA; further, the kit also comprises a single-stranded nucleic acid detector.

Further, the nucleic acid detection kit further comprises a primer for amplifying the target nucleic acid from the sample.

In another aspect, the invention also provides the use of the above buffer system suitable for the Cas12 protein in the preparation of a Cas12 protein-based nucleic acid detection kit.

In the present invention, the nucleic acid detection includes detection for viruses, bacteria, diseases, specific mutation sites or SNP sites; preferably, the virus is a plant virus or an animal virus; preferably, the virus is a coronavirus, preferably SARS, SARS-CoV2(COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-CoV. In the buffer system of the present invention, the Cas12 protein may exhibit Cis cleavage activity and/or Trans cleavage activity.

In one embodiment, the Cas12 protein includes, but is not limited to, one or any of Cas12a, Cas12b, Cas12i, Cas12 j;

in one embodiment, the amino acid sequence of Cas12i is shown as SEQ ID No.1, or a derivative protein formed by substitution, deletion or addition of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues of the amino acid sequence shown as SEQ ID No.1 or an active fragment thereof, and having substantially the same function.

In other embodiments, the amino acid sequence of Cas12j is as shown in SEQ ID No.2, or a derivative protein formed by substituting, deleting or adding one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues of the amino acid sequence shown in SEQ ID No.2 or an active fragment thereof, and having substantially the same function.

In other embodiments, the Cas12a is selected from one or any of FnCas12a, assas 12a, LbCas 12a, Lb5Cas12a, HkCas 12a, OsCas 12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12 a; the Cas12a is preferably LbCas 12a, the amino acid sequence is shown as SEQ ID No.3, or the derivative protein which is formed by substituting, deleting or adding one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues of the amino acid sequence shown as SEQ ID No.3 or an active fragment thereof and has basically the same function.

In other embodiments, the amino acid sequence of Cas12b is shown in SEQ ID No.4, or a derivative protein formed by substitution, deletion, or addition of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid residues of the amino acid sequence shown in SEQ ID No.4 or an active fragment thereof, and having substantially the same function.

In one embodiment, the Cas protein mutant comprises amino acid substitutions, deletions or substitutions, and the mutant retains at least its trans cleavage activity. Preferably, the mutant has Cis and trans cleavage activity.

General definition:

unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

As used herein, "pH buffer" refers to a solution formulated with a buffer pair consisting of "salts of a weak acid and its conjugate base" or "salts of a weak base and its conjugate acid" that is capable of slowing the change in pH upon the addition of a quantity of other substances. The pH buffer of the present invention is a pH buffer that maintains the pH of the solution at 7.0 to 9.0, preferably 7.6 to 8.5, more preferably 7.9. The preferred buffer herein is Tris-HCl buffer.

As used herein, "divalent cation" refers to a stable structure of atoms that lose 2 electrons, resulting in a positive charge. Common divalent cations are ferrous ions, copper ions, magnesium ions, and manganese ions. The most preferred divalent cation herein is magnesium.

As used herein, "DTT" also known as Dithiothreitol "or" Dithiothreitol "is a small organic reducing agent of formula C₄H₁₀O₂S₂. Are commonly used in solvents to stabilize enzymes and other proteins with free sulfhydryl groups.

As used herein, "BSA (Bovine Serum Albumin), also known as" Bovine Serum Albumin, "is a globulin in Bovine Serum comprising 583 amino acid residues, having a molecular weight of 66.5kDa and an isoelectric point of 4.7. BSA is generally used as a stabilizer in a preservation solution and a reaction solution for a restriction enzyme or a modification enzyme because some enzymes are unstable or have low activity at a low concentration. After BSA is added, it may play a role of "protection" or "carrier", and after many enzymes are added, the activity of BSA can be greatly improved.

As used herein, the "CRISPR" refers to clustered, regularly interspaced short palindromic repeats (clustered regularly interspaced short palindromic repeats) derived from the immune system of a microorganism.

Cas protein

As used herein, "Cas protein" refers to a CRISPR-associated protein, preferably a type V CRISPR/Cas protein, which, once bound to a target sequence (i.e., forming a ternary complex of Cas protein-gRNA-target sequence), can induce its trans activity, i.e., random cleavage of non-targeted single-stranded nucleotides (i.e., single-stranded nucleic acid detector, preferably single-stranded DNA (ssdna), single-stranded DNA-RNA hybrids, single-stranded RNA, as described herein). When the Cas protein is combined with the characteristic sequence, the protein can induce the trans activity by cutting or not cutting the characteristic sequence; preferably, it induces its trans activity by cleaving the signature sequence; more preferably, it induces its trans activity by cleaving the single-stranded signature sequence.

The Cas protein is a protein at least having trans cleavage activity, and preferably, the Cas protein is a protein having Cis and trans cleavage activity. The Cis activity refers to the activity that the Cas protein can recognize a PAM site and specifically cut a target sequence under the action of the gRNA.

Cas proteins described herein include Cas12 proteins, such as Cas12a, Cas12b, Cas12 d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12 j; preferably, the Cas protein is Cas12a, Cas12b, Cas12i, Cas12 j.

In embodiments, a Cas protein, as referred to herein, such as Cas12, also encompasses a functional variant of Cas or a homolog or ortholog thereof. As used herein, a "functional variant" of a protein refers to a variant of such a protein that at least partially retains the activity of the protein. Functional variants may include mutants (which may be insertion, deletion or substitution mutants), including polymorphs and the like. Also included in functional variants are fusion products of such proteins with another, usually unrelated, nucleic acid, protein, polypeptide or peptide. Functional variants may be naturally occurring or may be artificial. Advantageous embodiments may relate to engineered or non-naturally occurring V-type DNA targeting effector proteins.

In one embodiment, one or more nucleic acid molecules encoding a Cas protein, such as Cas12, or orthologs or homologs thereof, may be codon optimized for expression in a eukaryotic cell. Eukaryotes can be as described herein. One or more nucleic acid molecules may be engineered or non-naturally occurring.

In one embodiment, the Cas12 protein or ortholog or homolog thereof may comprise one or more mutations (and thus the nucleic acid molecule encoding it may have one or more mutations.

In one embodiment, the Cas protein may be from: cilium, listeria, corynebacterium, satrapia, legionella, treponema, Proteus, eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flavivivola, Flavobacterium, Azospirillum, Sphaerochaeta, gluconacetobacter, Neisseria, Rochelia, Parvibaculum, Staphylococcus, Nitrarefactor, Mycoplasma, Campylobacter, and Muspirillum.

gRNA

As used herein, the "gRNA" is also referred to as guide RNA or guide RNA and has a meaning commonly understood by those skilled in the art. In general, the guide RNA may comprise, or consist essentially of, a direct repeat and a guide sequence (guidessequence), also referred to as a spacer (spacer) in the context of an endogenous CRISPR system. grnas may include crRNA and tracrRNA or only crRNA depending on Cas protein on which they depend in different CRISPR systems. The crRNA and tracrRNA may be artificially engineered to fuse to form single guide RNA (sgRNA). In certain instances, the guide sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence (the signature sequence described in the present invention) to hybridize to the target sequence and direct specific binding of the CRISPR/Cas complex to the target sequence, typically having a sequence length of 12-25 nt. The direct repeat sequence can fold to form a specific structure (such as a stem-loop structure) for recognition by the Cas protein to form a complex. The targeting sequence need not be 100% complementary to the signature sequence (target sequence). The targeting sequence is not complementary to the single stranded nucleic acid detector.

In certain embodiments, the degree of complementarity (degree of match) between a targeting sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, when optimally aligned. Determining the optimal alignment is within the ability of one of ordinary skill in the art. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, the Smith-Waterman algorithm in matlab (Smith-Waterman), Bowtie, Geneius, Biopython, and SeqMan.

The gRNA of the invention can be natural, and can also be artificially modified or designed and synthesized.

The terms "polynucleotide", "nucleotide sequence", "nucleic acid molecule" and "nucleic acid" are used interchangeably and include DNA, RNA or hybrids thereof, whether double-stranded or single-stranded.

The term "homology" or "identity" is used to refer to a sequence between two polypeptides or between two nucleic acids

And (4) matching the situation. When two sequences to be compared are substituted by the same base or amino acid monomer at a position

When a subunit is occupied (e.g., a position in each of two DNA molecules is occupied by adenine,

or a position in each of the two polypeptides is occupied by a lysine), then the respective molecule is at that position

Are identical. Between the two sequences. Typically, this is done when the two sequences are aligned to yield maximum identity

And (6) comparing. Such an alignment can be determined by using, for example, the identity of the amino acid sequences by conventional methods, by computerized algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics package, Genetics, and Genetics Computer Group), with reference to, for example, the teachings of Smith and Waterman,1981, adv.appl.Math.2:482Pearson & Lipman,1988, Pro.Natl.Acad.Sci.USA85:244, Thompson et al, 1994, Nucleic Acids Res 22:467380, etc. The BLAST algorithm, available from the national center for Biotechnology information (NCBI www.ncbi.nlm.nih.gov /), can also be used, determined using default parameters.

Single-stranded nucleic acid detector

The single-stranded nucleic acid detector of the present invention is a sequence comprising 2 to 200 bases, preferably 2 to 150 bases, preferably 3 to 100 bases, preferably 3 to 30 bases, preferably 4 to 20 nucleobases, more preferably 5 to 15 bases. Preferably a single-stranded DNA molecule, a single-stranded RNA molecule or a single-stranded DNA-RNA hybrid.

The single-stranded nucleic acid detector is used in a detection method or system to report whether a characteristic sequence is contained. The single-stranded nucleic acid detector comprises different reporter groups or marker molecules at both ends, and does not present a reporter signal when in an initial state (i.e., an uncleaved state), and presents a detectable signal when the single-stranded nucleic acid detector is cleaved, i.e., presents a detectable difference after cleavage from before cleavage. In the present invention, if a detectable difference can be detected, it is reflected that the target nucleic acid contains a characteristic sequence to be detected; alternatively, if the detectable difference is not detectable, it indicates that the target nucleic acid does not contain the signature sequence to be detected.

In one embodiment, the reporter group or the marker molecule comprises a fluorescent group and a quenching group, wherein the fluorescent group is selected from one or any several of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

In one embodiment, the single stranded nucleic acid detector has a first molecule (e.g., FAM or FITC) attached to the 5 'end and a second molecule (e.g., biotin) attached to the 3' end. The reaction system containing the single-stranded nucleic acid detector is matched with the flow strip to detect the characteristic sequence (preferably, a colloidal gold detection mode). The flow strip is designed with two capture lines, with an antibody that binds to a first molecule (i.e. a first molecular antibody) at the sample contacting end (colloidal gold), an antibody that binds to the first molecular antibody at the first line (control line), and an antibody that binds to a second molecule (i.e. a second molecular antibody, such as avidin) at the second line (test line). As the reaction flows along the strip, the first molecular antibody binds to the first molecule carrying the cleaved or uncleaved oligonucleotide to the capture line, the cleaved reporter will bind to the antibody of the first molecular antibody at the first capture line, and the uncleaved reporter will bind to the second molecular antibody at the second capture line. Binding of the reporter group at each line will result in a strong readout/signal (e.g. color). As more reporters are cut, more signal will accumulate at the first capture line and less signal will appear at the second line. In certain aspects, the invention relates to the use of a flow strip as described herein for detecting nucleic acids. In certain aspects, the invention relates to a method of detecting nucleic acids using a flow strip as defined herein, e.g. a (side) flow test or a (side) flow immunochromatographic assay. In some aspects, the molecules in the single-stranded nucleic acid detector may be replaced with each other, or the positions of the molecules may be changed, and the modified form is also included in the present invention as long as the reporting principle is the same as or similar to that of the present invention.

Drawings

Figure 1. effect of different buffer systems on the sensitivity of Cas12i detection system. The lines are an experimental group of the buffer system 1, an experimental group of the buffer system 2, an experimental group of the buffer system 3, an experimental group of the buffer system 4 and a blank control.

Figure 2. effect of different concentrations of dtt (dithioreito) in buffer system components on the sensitivity of Cas12i detection system. The experimental group with the line (I) of 0mM DTT, the experimental group with the line (II) of 5mM DTT, the experimental group with the line (III) of 8mM DTT, the experimental group with the line (III) of 10mM DTT, the experimental group with the line (III) of 12mM DTT, the experimental group with the line (VI) of 15mM DTT, the experimental group with the line (III) of 20mM DTT and the line (III) of blank control.

FIG. 3 magnesium acetate (C) in the composition of the buffer system₄H₆O₄Mg) on the sensitivity of the Cas12i detection system, wherein line ① is the panel of 10mM magnesium acetate, line ② is the panel of 30mM magnesium acetate, line ③ is the panel of 50mM magnesium acetate, line ④ is the panel of 70mM magnesium acetate, line ⑤ is the panel of 90mM magnesium acetate, line ⑥ is the panel of 150mM magnesium acetate, line ⑦ is the panel of 200mM magnesium acetate, and line ⑧ is the blank.

Figure 4. effect of different concentrations of Tris-acetate in buffer system components on the sensitivity of Cas12i detection system. The test method comprises the following steps of firstly, obtaining a test group with a line of 0mM Tris-acetate, secondly, obtaining a test group with a line of 10mM Tris-acetate, thirdly, obtaining a test group with 20mM Tris-acetate, fourthly, obtaining a test group with 30mM Tris-acetate, fifthly, obtaining a test group with a line of 35mM Tris-acetate, sixthly, obtaining a test group with 40mM Tris-acetate, seventhly, obtaining a test group with 50mM Tris-acetate and controlling the line of eight.

Figure 5. effect of different concentrations of bsa (albumin from bone serum) in buffer system components on the sensitivity of Cas12i detection system. Wherein, the line (r) is an experimental group of 0 [ mu ] g/ml BSA, the line (r) is an experimental group of 50 [ mu ] g/ml BSA, the line (r) is an experimental group of 80 [ mu ] g/ml BSA, the line (r) is an experimental group of 100 [ mu ] g/ml BSA, the line (c) is an experimental group of 120 [ mu ] g/ml BSA, the line (c) is an experimental group of 150 [ mu ] g/ml BSA, the line (c) is an experimental group of 200 [ mu ] g/ml BSA, and the line (r) is a blank control.

Figure 6. effect of different pH of buffer system on the sensitivity of Cas12i detection system. Wherein, the line I is an experimental group with pH7.3, the line II is an experimental group with pH7.6, the line III is an experimental group with pH7.9, the line IV is an experimental group with pH8.2, the line IV is an experimental group with pH8.5, and the line IV is a blank control.

Figure 7 effect of different pH buffers on Cas12i detection system sensitivity. The three lines are an experiment group of Tris-acetic acid, an experiment group of Tris-HCl (Tris-hydrochloric acid), an experiment group of HEPES and a blank control.

Figure 8. effect of different reducing agents on the sensitivity of Cas12i detection system. Wherein, the line (I) is an experimental group of DTT, the line (II) is an experimental group of Tween-20, the line (III) is an experimental group of Triton X100, the line (III) is an experimental group of beta-mercaptoethanol, and the line (V) is blank control.

FIG. 9 shows the effect of different concentrations of beta-mercaptoethanol (2-Hydroxy-1-ethanethiol) in the buffer system components on the sensitivity of the Cas12i detection system. Wherein, the line (i) is an optimized experimental group of buffer 1, the line (ii) is an experimental group of 5mM beta-mercaptoethanol, the line (iii) is an experimental group of 10mM beta-mercaptoethanol, the line (iv) is an experimental group of 15mM beta-mercaptoethanol, the line (iv) is an experimental group of 20mM beta-mercaptoethanol, and the line (iv) is a blank control.

Figure 10 effect of different magnesium salts on the sensitivity of Cas12i detection system. Wherein, the line I is a control group of magnesium acetate, the line II is a control group of magnesium sulfate, the line III is a control group of magnesium chloride, and the line IV is a blank control.

Fig. 11. effect of optimized buffer system 1 and buffer system 1 on Cas12i detection system sensitivity. The line (i) is an experimental group of the optimized buffer system 1, the line (ii) is an experimental group of the buffer system 1, and the line (iii) is a blank control.

Fig. 12 detection sensitivity of Cas12a, Cas12b, and Cas12i in optimized buffer system 1. The line (i) is an experimental group of Cas12i, the line (ii) is an experimental group of Cas12a, the line (iii) is an experimental group of Cas12b, and the line (iv) is a blank control.

Detailed description of the preferred embodiments

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

In this embodiment, the Cas12 protein is used for target nucleic acid detection based on the following principle: guiding the Cas protein to recognize and bind to the target nucleic acid using a gRNA that can pair with the target nucleic acid; subsequently, the Cas protein activates trans cleavage activity, cleaving the single-stranded nucleic acid detector; the two ends of the single-stranded nucleic acid detector are respectively provided with a fluorescent group and a quenching group, and if the single-stranded nucleic acid detector is cut, fluorescence can be excited; in other embodiments, both ends of the single-stranded nucleic acid detector may be provided with a label capable of being detected by colloidal gold.

Example 1 effect of different buffer systems on the sensitivity of Cas12i detection system.

Different buffer systems were formulated, the composition of each buffer system being shown in table 1:

	buffer system 1	Buffer system 2	Buffer system 3	Buffer system 4
					Tris-acetic acid (mM)	36			20
Tris-hydrochloric acid (mM)		10	20
					Magnesium acetate (mM)	10		3.5	10
Potassium acetate (mM)				100
					BSA(μg/ml)	100	100
DTT(mM)	10			1
					Sodium chloride (mM)		50	40
Potassium chloride (mM)			10
					Magnesium chloride (mM)		10
pH value	7.8	7.9	7.8	7.8

TABLE 1 composition of the respective buffer systems

Cas12i, gRNA, ssDNA (target nucleic acid sequence single-stranded DNA) and Reporter (5 '-FAM-TTGTT-3' BHQ) were added to each buffer system, respectively, with a final concentration of Cas12i of 100nM, a final concentration of gRNA of 50nM, a final concentration of ssDNA (target nucleic acid sequence single-stranded DNA) of 500nM, and a final concentration of Reporter (5 '-FAM-TTGTT-3' BHQ) of 500 nM.

As a result, as shown in fig. 1, in

buffer systems

1 and 2, Cas12i can express higher sensitivity, and a significant fluorescence signal can be detected in about 5 minutes; in buffer systems 3 and 4, Cas12i has lower detection sensitivity.

Example 2 Effect of different concentrations of DTT (dithioreito) on the sensitivity of Cas12i detection System

A series of buffer systems were prepared, all containing 36mM Tris-acetate, 10mM magnesium acetate, 100. mu.g/ml BSA (same as buffer system 1). Except that the final concentrations of DTT were 0mM, 5mM, 8mM, 10mM, 12mM, 15mM, and 20mM, respectively.

As shown in FIG. 2, fluorescence signals were detected rapidly at a final concentration of 5-20mM DTT. Wherein, the sensitivity of fluorescence detection is highest when the final concentration of DTT is 12 mM.

Example 3 magnesium acetate (C) at various concentrations₄H₆O₄Mg) effect on Cas12i detection system sensitivity

A series of buffer systems were prepared, all containing 36mM Tris-acetate, 10mM DTT, 100. mu.g/ml BSA (same as buffer system 1). The final concentrations of magnesium acetate were 10mM, 30mM, 50mM, 70mM, 90mM, 150mM, and 200mM, respectively.

As shown in FIG. 3, the fluorescence signal was detected within 3 minutes at a final concentration of 0-200mM magnesium acetate, and the fluorescence detection sensitivity was high at a final concentration of 30-150mM magnesium acetate.

Example 4 Effect of varying concentrations of Tris-acetate (Tris-acetate) on the sensitivity of Cas12i detection systems

A series of buffer systems were prepared, all containing 10mM magnesium acetate, 100. mu.g/ml BSA and 10mM DTT (same as buffer system 1). With the exception that the final concentration of Tris-acetate was 0mM, 10mM, 20mM, 30mM, 35mM, 40mM, 50mM, respectively.

As a result, as shown in FIG. 4, fluorescence was rapidly exhibited in the buffer system containing Tris-acetate.

Example 5 Effect of different concentrations of BSA (Albumin from bone serum) on the sensitivity of Cas12i detection System

A series of buffer systems were prepared, all containing 10mM magnesium acetate, 35mM Tris-acetate and 10mM DTT (same as buffer system 1). Except that the final concentrations of BSA were 0. mu.g/ml, 50. mu.g/ml, 80. mu.g/ml, 100. mu.g/ml, 120. mu.g/ml, 150. mu.g/ml, 200. mu.g/ml, respectively.

As shown in FIG. 5, fluorescence was rapidly reported at a final BSA concentration of 50-200. mu.g/ml, and the sensitivity was highest at a final BSA concentration of 80-120. mu.g/ml.

Example 6 Effect of different pH on Cas12i detection System sensitivity

A series of buffer systems were prepared, all containing 36mM Tris-acetate, 10mM magnesium acetate, 10mM DTT, 100. mu.g/ml BSA (same as buffer system 1). The pH of the solution was 7.3, 7.6, 7.9, 8.2, 8.5, respectively.

As a result, as shown in FIG. 6, the fluorescence detection sensitivity was higher at pH in the range of 7.6 to 8.5 than at other concentrations.

Example 7 Effect of different buffer systems on the sensitivity of Cas12i detection System

In this embodiment, the final concentration of Cas12i, gRNA, ssDNA (single-stranded DNA of the target nucleic acid sequence), Reporter (5 '-FAM-TTGTT-3' BHQ), magnesium acetate, BSA, and DTT in all systems was 100nM, 50nM, 500nM, and 12mM, respectively.

Except that the pH buffer of the experimental group 1 was Tris-acetate, and the fluorescence detection signal was shown as line (r) in FIG. 7; the pH buffer of the experimental group 2 is Tris-HCl, and the fluorescence detection signal is shown as the line II in FIG. 7; the pH buffer of the experimental group 3 is HEPES, and the fluorescence detection signal is shown as the line (c) of FIG. 7; line iv is the blank control. The final concentration of all pH buffers was 40 mM.

As a result, as shown in fig. 7, Cas12i can rapidly report fluorescence in the above buffer; among them, Cas12i detection sensitivity was highest in Tris-acetate (Tris-OAC) buffer system.

Example 8 Effect of different reducing Agents on Cas12i detection System sensitivity

In this embodiment, the final concentration of Cas12i, gRNA, ssDNA (single-stranded DNA of the target nucleic acid sequence), Reporter (5 '-FAM-TTGTT-3' BHQ), magnesium acetate, BSA, and Tris-acetic acid in all systems was 100nM, 50nM, 500nM, and 500 mM, respectively, and the final concentration of Reporter (5 '-FAM-TTGTT-3' BHQ), 30mM, 120. mu.g/ml, and 40mM, respectively.

Except that the reducing agent of the experimental group 1 was DTT at 12mM, and the fluorescence detection signal was shown as line (r) in FIG. 8; the reducing agent of the experimental group 2 is 0.1 percent of Tween-20, and the fluorescence detection signal is shown as a line II in FIG. 8; the reducing agent of the experimental group 3 is 0.1% of Triton X100, and the fluorescence detection signal is shown as a line (c) in FIG. 8; the reducing agent of the experimental group 4 is 1mM beta-mercaptoethanol, and the fluorescence detection signal is shown as a line (r) in FIG. 8; line (v) is blank control.

The results are shown in fig. 8, the detection sensitivity of Cas12i is highest when DTT is a reducing agent, the detection sensitivity of Cas12i is general when β -mercaptoethanol is a reducing agent, and the detection sensitivity of Cas12i is poor when Tween-20 and Triton X100 are reducing agents.

Example 9 influence of different concentrations of beta-mercaptoethanol (2-Hydroxy-1-ethanethiol) on the sensitivity of Cas12i detection system

A series of buffer systems were prepared, all containing 40mM Tris-acetate, 30mM magnesium acetate, 120. mu.g/ml BSA. Except that the final concentrations of beta-mercaptoethanol (2-Hydroxy-1-ethanethiol) were 5mM, 10mM, 15mM, and 20mM, respectively.

As shown in FIG. 9, the fluorescence signal was rapidly detected at the final concentration of 10-20mM β -mercaptoethanol.

Example 10 Effect of different magnesium salts on the sensitivity of Cas12i detection System

In this embodiment, the final concentration of Cas12i, gRNA, ssDNA (single-stranded DNA of the target nucleic acid sequence), Reporter (5 '-FAM-TTGTT-3' BHQ), BSA, Tris-acetic acid and DTT in all systems was 100nM, 50nM, 500nM, 120. mu.g/ml, 40mM and 12mM, respectively.

Except that the magnesium salt of experiment group 1 was magnesium acetate, the fluorescence detection signal was as shown by line ① in FIG. 9, and the magnesium salt of experiment group 2 was MgSO₄The fluorescence signal is shown as line ② in FIG. 9, the magnesium salt in group 3 is MgCl, the fluorescence signal is shown as line ③ in FIG. 9, line ④ is blank, and the final concentration of all magnesium salts is 30 mM.

The results are shown in fig. 10, with different magnesium salts having little effect on Cas12i detection sensitivity.

Example 11 influence of optimized buffer System 1 and non-optimized buffer System 1 on Cas12i detection sensitivity

An optimized buffer system 1 and a buffer system 1 are prepared, wherein the pH of the buffer is shown as the components in the following table 2:

	optimized damping system 1	Buffer system 1
			Tris-acetic acid (mM)	40	36
Magnesium acetate (mM)	30	10
			BSA(μg/ml)	120	100
DTT(mM)	12	10
			pH	7.9	7.8

TABLE 2 optimized buffer System 1 and composition of buffer System 1

As a result, as shown in fig. 11, Cas12i detection sensitivity was significantly improved in the optimized buffer system 1 compared to the buffer system 1 before optimization.

Example 12 Effect of optimized buffer System 1 on Cas12a, Cas12b and Cas12i detection sensitivity

Optimized buffer system 1 was formulated, and Cas12a, Cas12b and Cas12i, Cas12a, Cas12b and Cas12i were added to each reaction system, respectively, at a final concentration of 100 nM. In addition, gRNA, ssDNA (target nucleic acid sequence single-stranded DNA), and Reporter (5 '-FAM-TTGTT-3' BHQ) were added to each reaction system, respectively, with a final concentration of 50nM for gRNA, 500nM for ssDNA (target nucleic acid sequence single-stranded DNA), and 500nM for Reporter (5 '-FAM-TTGTT-3' BHQ).

As a result, as shown in fig. 12, in the optimized buffer system 1, Cas12i, Cas12a, and Cas12b all have higher detection sensitivity.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shunheng Biotech Co., Ltd

<120> buffer system applicable to Cas12 protein and application thereof

<130>P2020-1396

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>1045

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>1

Met Lys Lys Val Glu Val Ser Arg Pro Tyr Gln Ser Leu Leu Leu Pro

1 5 10 15

Asn His Arg Lys Phe Lys Tyr Leu Asp Glu Thr Trp Asn Ala Tyr Lys

2025 30

Ser Val Lys Ser Leu Leu His Arg Phe Leu Val Cys Ala Tyr Gly Ala

35 40 45

Val Pro Phe Asn Lys Phe Val Glu Val Val Glu Lys Val Asp Asn Asp

50 55 60

Gln Leu Val Leu Ala Phe Ala Val Arg Leu Phe Arg Leu Val Pro Val

65 70 75 80

Glu Ser Thr Ser Phe Ala Lys Val Asp Lys Ala Asn Leu Ala Lys Ser

85 90 95

Leu Ala Asn His Leu Pro Val Gly Thr Ala Ile Pro Ala Asn Val Gln

100 105 110

Ser Tyr Phe Asp Ser Asn Phe Asp Pro Lys Lys Tyr Met Trp Ile Asp

115 120 125

Cys Ala Trp Glu Ala Asp Arg Leu Ala Arg Glu Met Gly Leu Ser Ala

130 135 140

Ser Gln Phe Ser Glu Tyr Ala Thr Thr Met Leu Trp Glu Asp Trp Leu

145 150 155 160

Pro Leu Asn Lys Asp Asp Val Asn Gly Trp Gly Ser Val Ser Gly Leu

165 170 175

Phe Gly Glu Gly Lys Lys Glu Asp Arg Gln Gln Lys Val Lys Met Leu

180 185190

Asn Asn Leu Leu Asn Gly Ile Lys Lys Asn Pro Pro Lys Asp Tyr Thr

195 200 205

Gln Tyr Leu Lys Ile Leu Leu Asn Ala Phe Asp Ala Lys Ser His Lys

210 215 220

Glu Ala Val Lys Asn Tyr Lys Gly Asp Ser Thr Gly Arg Thr Ala Ser

225 230 235 240

Tyr Leu Ser Glu Lys Ser Gly Glu Ile Thr Glu Leu Met Leu Glu Gln

245 250 255

Leu Met Ser Asn Ile Gln Arg Asp Ile Gly Asp Lys Gln Lys Glu Ile

260 265 270

Ser Leu Pro Lys Lys Asp Val Val Lys Lys Tyr Leu Glu Ser Glu Ser

275 280 285

Gly Val Pro Tyr Asp Gln Asn Leu Trp Ser Gln Ala Tyr Arg Asn Ala

290 295 300

Ala Ser Ser Ile Lys Lys Thr Asp Thr Arg Asn Phe Asn Ser Thr Leu

305 310 315 320

Glu Lys Phe Lys Asn Glu Val Glu Leu Arg Gly Leu Leu Ser Glu Gly

325 330 335

Asp Asp Val Glu Ile Leu Arg Ser Lys Phe Phe Ser Ser Glu Phe His

340 345350

Lys Thr Pro Asp Lys Phe Val Ile Lys Pro Glu His Ile Gly Phe Asn

355 360 365

Asn Lys Tyr Asn Val Val Ala Glu Leu Tyr Lys Leu Lys Ala Glu Ala

370 375 380

Thr Asp Phe Glu Ser Ala Phe Ala Thr Val Lys Asp Glu Phe Glu Glu

385 390 395 400

Lys Gly Ile Lys His Pro Ile Lys Asn Ile Leu Glu Tyr Ile Trp Asn

405 410 415

Asn Glu Val Pro Val Glu Lys Trp Gly Arg Val Ala Arg Phe Asn Gln

420 425 430

Ser Glu Glu Lys Leu Leu Arg Ile Lys Ala Asn Pro Thr Val Glu Cys

435 440 445

Asn Gln Gly Met Thr Phe Gly Asn Ser Ala Met Val Gly Glu Val Leu

450 455 460

Arg Ser Asn Tyr Val Ser Lys Lys Gly Ala Leu Val Ser Gly Glu His

465 470 475 480

Gly Gly Arg Leu Ile Gly Gln Asn Asn Met Ile Trp Leu Glu Met Arg

485 490 495

Leu Leu Asn Lys Gly Lys Trp Glu Thr His His Val Pro Thr His Asn

500 505 510

Met Lys Phe Phe Glu Glu Val His Ala Tyr Asn Pro Ser Leu Ala Asp

515 520 525

Ser Val Asn Val Arg Asn Arg Leu Tyr Arg Ser Glu Asp Tyr Thr Gln

530 535 540

Leu Pro Ser Ser Ile Thr Asp Gly Leu Lys Gly Asn Pro Lys Ala Lys

545 550 555 560

Leu Leu Lys Arg Gln His Cys Ala Leu Asn Asn Met Thr Ala Asn Val

565 570 575

Leu Asn Pro Lys Leu Ser Phe Thr Ile Asn Lys Lys Asn Asp Asp Tyr

580 585 590

Thr Val Ile Ile Val His Ser Val Glu Val Ser Lys Pro Arg Arg Glu

595 600 605

Val Leu Val Gly Asp Tyr Leu Val Gly Met Asp Gln Asn Gln Thr Ala

610 615 620

Ser Asn Thr Tyr Ala Val Met Gln Val Val Lys Pro Lys Ser Thr Asp

625 630 635 640

Ala Ile Pro Phe Arg Asn Met Trp Val Arg Phe Val Glu Ser Gly Ser

645 650 655

Ile Glu Ser Arg Thr Leu Asn Ser Arg Gly Glu Tyr Val Asp Gln Leu

660 665 670

Asn His Asp Gly Val Asp Leu Phe Glu Ile Gly Asp Thr Glu Trp Val

675 680 685

Asp Ser Ala Arg Lys Phe Phe Asn Lys Leu Gly Val Lys His Lys Asp

690 695 700

Gly Thr Leu Val Asp Leu Ser Thr Ala Pro Arg Lys Ala Tyr Ala Phe

705 710 715 720

Asn Asn Phe Tyr Phe Lys Thr Met Leu Asn His Leu Arg Ser Asn Glu

725 730 735

Val Asp Leu Thr Leu Leu Arg Asn Glu Ile Leu Arg Val Ala Asn Gly

740 745 750

Arg Phe Ser Pro Met Arg Leu Gly Ser Leu Ser Trp Thr Thr Leu Lys

755 760 765

Ala Leu Gly Ser Phe Lys Ser Leu Val Leu Ser Tyr Phe Asp Arg Leu

770 775 780

Gly Ala Lys Glu Met Val Asp Lys Glu Ala Lys Asp Lys Ser Leu Phe

785 790 795 800

Asp Leu Leu Val Ala Ile Asn Asn Lys Arg Ser Asn Lys Arg Glu Glu

805 810 815

Arg Thr Ser Arg Ile Ala Ser Ser Leu Met Thr Val Ala Gln Lys Tyr

820 825 830

Lys Val Asp Asn Ala Val Val His Val Val Val Glu Gly Asn Leu Ser

835 840 845

Ser Thr Asp Arg Ser Ala Ser Lys Ala His Asn Arg Asn Thr Met Asp

850 855 860

Trp Cys Ser Arg Ala Val Val Lys Lys Leu Glu Asp Met Cys Asn Leu

865 870 875 880

Tyr Gly Phe Asn Ile Lys Gly Val Pro Ala Phe Tyr Thr Ser His Gln

885 890 895

Asp Pro Leu Val His Arg Ala Asp Tyr Asp Asp Pro Lys Pro Ala Leu

900 905 910

Arg Cys Arg Tyr Ser Ser Tyr Ser Arg Ala Asp Phe Ser Lys Trp Gly

915 920 925

Gln Asn Ala Leu Ala Ala Val Val Arg Trp Ala Ser Asn Lys Lys Ser

930 935 940

Asn Thr Cys Tyr Lys Val Gly Ala Val Glu Phe Leu Lys Gln His Gly

945 950 955 960

Leu Phe Ala Asp Lys Lys Leu Thr Val Glu Gln Phe Leu Ser Lys Val

965 970 975

Lys Asp Glu Glu Ile Leu Ile Pro Arg Arg Gly Gly Arg Val Phe Leu

980 985 990

Thr Thr His Arg Leu Leu Ala Glu Ser Thr Phe Val Tyr Leu Asn Gly

995 1000 1005

Val Lys Tyr His Ser Cys Asn Ala Asp Glu Val Ala Ala Val Asn Ile

1010 1015 1020

Cys Leu Asn Asp Trp Val Ile Pro Cys Lys Lys Lys Met Lys Glu Glu

1025 1030 1035 1040

Ser Ser Ala Ser Gly

1045

<210>2

<211>908

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>2

Met Pro Ser Tyr Lys Ser Ser Arg Val Leu Val Arg Asp Val Pro Glu

1 5 10 15

Glu Leu Val Asp His Tyr Glu Arg Ser His Arg Val Ala Ala Phe Phe

20 25 30

Met Arg Leu Leu Leu Ala Met Arg Arg Glu Pro Tyr Ser Leu Arg Met

35 40 45

Arg Asp Gly Thr Glu Arg Glu Val Asp Leu Asp Glu Thr Asp Asp Phe

50 55 60

Leu Arg Ser Ala Gly Cys Glu Glu Pro Asp Ala Val Ser Asp Asp Leu

65 70 75 80

Arg Ser Phe Ala Leu Ala Val Leu His Gln Asp Asn Pro Lys Lys Arg

85 90 95

Ala Phe Leu Glu Ser Glu Asn Cys Val Ser Ile Leu Cys Leu Glu Lys

100 105 110

Ser Ala Ser Gly Thr Arg Tyr Tyr Lys Arg Pro Gly Tyr Gln Leu Leu

115 120 125

Lys Lys Ala Ile Glu Glu Glu Trp Gly Trp Asp Lys Phe Glu Ala Ser

130 135 140

Leu Leu Asp Glu Arg Thr Gly Glu Val Ala Glu Lys Phe Ala Ala Leu

145 150 155 160

Ser Met Glu Asp Trp Arg Arg Phe Phe Ala Ala Arg Asp Pro Asp Asp

165 170 175

Leu Gly Arg Glu Leu Leu Lys Thr Asp Thr Arg Glu Gly Met Ala Ala

180 185 190

Ala Leu Arg Leu Arg Glu Arg Gly Val Phe Pro Val Ser Val Pro Glu

195 200 205

His Leu Asp Leu Asp Ser Leu Lys Ala Ala Met Ala Ser Ala Ala Glu

210 215 220

Arg Leu Lys Ser Trp Leu Ala Cys Asn Gln Arg Ala Val Asp Glu Lys

225 230 235 240

Ser Glu Leu Arg Lys Arg Phe Glu Glu Ala Leu Asp Gly Val Asp Pro

245 250 255

Glu Lys Tyr Ala Leu Phe Glu Lys Phe Ala Ala Glu Leu Gln Gln Ala

260 265 270

Asp Tyr Asn Val Thr Lys Lys Leu Val Leu Ala Val Ser Ala Lys Phe

275 280 285

Pro Ala Thr Glu Pro Ser Glu Phe Lys Arg Gly Val Glu Ile Leu Lys

290 295 300

Glu Asp Gly Tyr Lys Pro Leu Trp Glu Asp Phe Arg Glu Leu Gly Phe

305 310 315 320

Val Tyr Leu Ala Glu Arg Lys Trp Glu Arg Arg Arg Gly Gly Ala Ala

325 330 335

Val Thr Leu Cys Asp Ala Asp Asp Ser Pro Ile Lys Val Arg Phe Gly

340 345 350

Leu Thr Gly Arg Gly Arg Lys Phe Val Leu Ser Ala Ala Gly Ser Arg

355 360 365

Phe Leu Ile Thr Val Lys Leu Pro Cys Gly Asp Val Gly Leu Thr Ala

370 375 380

Val Pro Ser Arg Tyr Phe Trp Asn Pro Ser Val Gly Arg Thr Thr Ser

385 390 395 400

Asn Ser Phe Arg Ile Glu Phe Thr Lys Arg Thr Thr Glu Asn Arg Arg

405 410 415

Tyr Val Gly Glu Val Lys Glu Ile Gly Leu Val Arg Gln Arg Gly Arg

420 425 430

Tyr Tyr Phe Phe Ile Asp Tyr Asn Phe Asp Pro Glu Glu Val Ser Asp

435 440 445

Glu Thr Lys Val Gly Arg Ala Phe Phe Arg Ala Pro Leu Asn Glu Ser

450 455 460

Arg Pro Lys Pro Lys Asp Lys Leu Thr Val Met Gly Ile Asp Leu Gly

465 470 475 480

Ile Asn Pro Ala Phe Ala Phe Ala Val Cys Thr Leu Gly Glu Cys Gln

485 490 495

Asp Gly Ile Arg Ser Pro Val Ala Lys Met Glu Asp Val Ser Phe Asp

500 505 510

Ser Thr Gly Leu Arg Gly Gly Ile Gly Ser Gln Lys Leu His Arg Glu

515 520 525

Met His Asn Leu Ser Asp Arg Cys Phe Tyr Gly Ala Arg Tyr Ile Arg

530 535 540

Leu Ser Lys Lys Leu Arg Asp Arg Gly Ala Leu Asn Asp Ile Glu Ala

545 550 555 560

Arg Leu Leu Glu Glu Lys Tyr Ile Pro Gly Phe Arg Ile Val His Ile

565 570 575

Glu Asp Ala Asp Glu Arg Arg Arg Thr Val Gly Arg Thr Val Lys Glu

580 585 590

Ile Lys Gln Glu Tyr Lys Arg Ile Arg His Gln Phe Tyr Leu Arg Tyr

595 600 605

His Thr Ser Lys Arg Asp Arg Thr Glu Leu Ile Ser Ala Glu Tyr Phe

610 615 620

Arg Met Leu Phe Leu Val Lys Asn Leu Arg Asn Leu Leu Lys Ser Trp

625 630 635 640

Asn Arg Tyr His Trp Thr Thr Gly Asp Arg Glu Arg Arg Gly Gly Asn

645 650 655

Pro Asp Glu Leu Lys Ser Tyr Val Arg Tyr Tyr Asn Asn Leu Arg Met

660 665 670

Asp Thr Leu Lys Lys Leu Thr Cys Ala Ile Val Arg Thr Ala Lys Glu

675 680 685

His Gly Ala Thr Leu Val Ala Met Glu Asn Ile Gln Arg Val Asp Arg

690 695 700

Asp Asp Glu Val Lys Arg Arg Lys Glu Asn Ser Leu Leu Ser Leu Trp

705 710 715 720

Ala Pro Gly Met Val Leu Glu Arg Val Glu Gln Glu Leu Lys Asn Glu

725 730 735

Gly Ile Leu Ala Trp Glu Val Asp Pro Arg His Thr Ser Gln Thr Ser

740 745 750

Cys Ile Thr Asp Glu Phe Gly Tyr Arg Ser Leu Val Ala Lys Asp Thr

755 760 765

Phe Tyr Phe Glu Gln Asp Arg Lys Ile His Arg Ile Asp Ala Asp Val

770 775 780

Asn Ala Ala Ile Asn Ile Ala Arg Arg Phe Leu Thr Arg Tyr Arg Ser

785 790 795 800

Leu Thr Gln Leu Trp Ala Ser Leu Leu Asp Asp Gly Arg Tyr Leu Val

805 810 815

Asn Val Thr Arg Gln His Glu Arg Ala Tyr Leu Glu Leu Gln Thr Gly

820 825 830

Ala Pro Ala Ala Thr Leu Asn Pro Thr Ala Glu Ala Ser Tyr Glu Leu

835 840 845

Val Gly Leu Ser Pro Glu Glu Glu Glu Leu Ala Gln Thr Arg Ile Lys

850 855 860

Arg Lys Lys Arg Glu Pro Phe Tyr Arg His Glu Gly Val Trp Leu Thr

865 870 875 880

Arg Glu Lys His Arg Glu Gln Val His Glu Leu Arg Asn Gln Val Leu

885 890 895

Ala Leu Gly Asn Ala Lys Ile Pro Glu Ile Arg Thr

900 905

<210>3

<211>1228

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>3

Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp

20 25 30

Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys

35 40 45

Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp

50 55 60

Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu

65 70 75 80

Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn

85 90 95

Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn

100105 110

Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu

115 120 125

Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe

130 135 140

Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn

145 150 155 160

Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile

165 170 175

Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys

180 185 190

Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys

195 200 205

Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe

210 215 220

Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile

225 230 235 240

Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn

245 250 255

Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys

260265 270

Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser

275 280 285

Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe

290 295 300

Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys

305 310 315 320

Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile

325 330 335

Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe

340 345 350

Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp

355 360 365

Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp

370 375 380

Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu

385 390 395 400

Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu

405 410 415

Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser

420 425430

Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys

435 440 445

Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys

450 455 460

Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr

465 470 475 480

Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile

485 490 495

Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr

500 505 510

Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro

515 520 525

Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala

530 535 540

Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys

545 550 555 560

Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly

565 570 575

Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met

580 585590

Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro

595 600 605

Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly

610 615 620

Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys

625 630 635 640

Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn

645 650 655

Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu

660 665 670

Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys

675 680 685

Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile

690 695 700

Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His

705 710 715 720

Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile

725 730 735

Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys

740 745 750

Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys

755 760 765

Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr

770 775 780

Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile

785 790 795 800

Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val

805 810 815

Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp

820 825 830

Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly

835 840 845

Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn

850 855 860

Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu

865 870 875 880

Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile

885 890 895

Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys

900 905 910

Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn

915 920 925

Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln

930 935 940

Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys

945 950 955 960

Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile

965 970 975

Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe

980 985 990

Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr

995 1000 1005

Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp Ser

1010 1015 1020

Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu Glu

1025 1030 1035 1040

Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr Asp

1045 1050 1055

Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr Gly Asn Arg Ile

1060 1065 1070

Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val Phe Asp Trp Glu Glu

1075 1080 1085

Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys Tyr Gly Ile

1090 1095 1100

Asn Tyr Gln Gln Gly Asp Ile Arg Ala Leu Leu Cys Glu Gln Ser Asp

1105 1110 1115 1120

Lys Ala Phe Tyr Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu Gln

1125 1130 1135

Met Arg Asn Ser Ile Thr Gly Arg Thr Asp Val Asp Phe Leu Ile Ser

1140 1145 1150

Pro Val Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu

1155 1160 1165

Ala Gln Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala

1170 1175 1180

Tyr Asn Ile Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys

1185 1190 1195 1200

Ala Glu Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys

1205 1210 1215

Glu Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys His

1220 1225

<210>4

<211>1129

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>4

Met Ala Val Lys Ser Ile Lys Val Lys Leu Arg Leu Asp Asp Met Pro

1 5 10 15

Glu Ile Arg Ala Gly Leu Trp Lys Leu His Lys Glu Val Asn Ala Gly

20 25 30

Val Arg Tyr Tyr Thr Glu Trp Leu Ser Leu Leu Arg Gln Glu Asn Leu

35 40 45

Tyr Arg Arg Ser Pro Asn Gly Asp Gly Glu Gln Glu Cys Asp Lys Thr

50 55 60

Ala Glu Glu Cys Lys Ala Glu Leu Leu Glu Arg Leu Arg Ala Arg Gln

65 70 75 80

Val Glu Asn Gly His Arg Gly Pro Ala Gly Ser Asp Asp Glu Leu Leu

85 90 95

Gln Leu Ala Arg Gln Leu Tyr Glu Leu Leu Val Pro Gln Ala Ile Gly

100 105 110

Ala Lys Gly Asp Ala Gln Gln Ile Ala Arg Lys Phe Leu Ser Pro Leu

115 120 125

Ala Asp Lys Asp Ala Val Gly Gly Leu Gly Ile Ala Lys Ala Gly Asn

130 135 140

Lys Pro Arg Trp Val Arg Met Arg Glu Ala Gly Glu Pro Gly Trp Glu

145 150 155 160

Glu Glu Lys Glu Lys Ala Glu Thr Arg Lys Ser Ala Asp Arg Thr Ala

165 170 175

Asp Val Leu Arg Ala Leu Ala Asp Phe Gly Leu Lys Pro Leu Met Arg

180 185 190

Val Tyr Thr Asp Ser Glu Met Ser Ser Val Glu Trp Lys Pro Leu Arg

195 200 205

Lys Gly Gln Ala Val Arg Thr Trp Asp Arg Asp Met Phe Gln Gln Ala

210 215 220

Ile Glu Arg Met Met Ser Trp Glu Ser Trp Asn Gln Arg Val Gly Gln

225 230 235 240

Glu Tyr Ala Lys Leu Val Glu Gln Lys Asn Arg Phe Glu Gln Lys Asn

245 250 255

Phe Val Gly Gln Glu His Leu Val His Leu Val Asn Gln Leu Gln Gln

260 265 270

Asp Met Lys Glu Ala Ser Pro Gly Leu Glu Ser Lys Glu Gln Thr Ala

275 280 285

His Tyr Val Thr Gly Arg Ala Leu Arg Gly Ser Asp Lys Val Phe Glu

290295 300

Lys Trp Gly Lys Leu Ala Pro Asp Ala Pro Phe Asp Leu Tyr Asp Ala

305 310 315 320

Glu Ile Lys Asn Val Gln Arg Arg Asn Thr Arg Arg Phe Gly Ser His

325 330 335

Asp Leu Phe Ala Lys Leu Ala Glu Pro Glu Tyr Gln Ala Leu Trp Arg

340 345 350

Glu Asp Ala Ser Phe Leu Thr Arg Tyr Ala Val Tyr Asn Ser Ile Leu

355 360 365

Arg Lys Leu Asn His Ala Lys Met Phe Ala Thr Phe Thr Leu Pro Asp

370 375 380

Ala Thr Ala His Pro Ile Trp Thr Arg Phe Asp Lys Leu Gly Gly Asn

385 390 395 400

Leu His Gln Tyr Thr Phe Leu Phe Asn Glu Phe Gly Glu Arg Arg His

405 410 415

Ala Ile Arg Phe His Lys Leu Leu Lys Val Glu Asn Gly Val Ala Arg

420 425 430

Glu Val Asp Asp Val Thr Val Pro Ile Ser Met Ser Glu Gln Leu Asp

435 440 445

Asn Leu Leu Pro Arg Asp Pro Asn Glu Pro Ile Ala Leu Tyr Phe Arg

450455 460

Asp Tyr Gly Ala Glu Gln His Phe Thr Gly Glu Phe Gly Gly Ala Lys

465 470 475 480

Ile Gln Cys Arg Arg Asp Gln Leu Ala His Met His Arg Arg Arg Gly

485 490 495

Ala Arg Asp Val Tyr Leu Asn Val Ser Val Arg Val Gln Ser Gln Ser

500 505 510

Glu Ala Arg Gly Glu Arg Arg Pro Pro Tyr Ala Ala Val Phe Arg Leu

515 520 525

Val Gly Asp Asn His Arg Ala Phe Val His Phe Asp Lys Leu Ser Asp

530 535 540

Tyr Leu Ala Glu His Pro Asp Asp Gly Lys Leu Gly Ser Glu Gly Leu

545 550 555 560

Leu Ser Gly Leu Arg Val Met Ser Val Asp Leu Gly Leu Arg Thr Ser

565 570 575

Ala Ser Ile Ser Val Phe Arg Val Ala Arg Lys Asp Glu Leu Lys Pro

580 585 590

Asn Ser Lys Gly Arg Val Pro Phe Phe Phe Pro Ile Lys Gly Asn Asp

595 600 605

Asn Leu Val Ala Val His Glu Arg Ser Gln Leu Leu Lys Leu Pro Gly

610615 620

Glu Thr Glu Ser Lys Asp Leu Arg Ala Ile Arg Glu Glu Arg Gln Arg

625 630 635 640

Thr Leu Arg Gln Leu Arg Thr Gln Leu Ala Tyr Leu Arg Leu Leu Val

645 650 655

Arg Cys Gly Ser Glu Asp Val Gly Arg Arg Glu Arg Ser Trp Ala Lys

660 665 670

Leu Ile Glu Gln Pro Val Asp Ala Ala Asn His Met Thr Pro Asp Trp

675 680 685

Arg Glu Ala Phe Glu Asn Glu Leu Gln Lys Leu Lys Ser Leu His Gly

690 695 700

Ile Cys Ser Asp Lys Glu Trp Met Asp Ala Val Tyr Glu Ser Val Arg

705 710 715 720

Arg Val Trp Arg His Met Gly Lys Gln Val Arg Asp Trp Arg Lys Asp

725 730 735

Val Arg Ser Gly Glu Arg Pro Lys Ile Arg Gly Tyr Ala Lys Asp Val

740 745 750

Val Gly Gly Asn Ser Ile Glu Gln Ile Glu Tyr Leu Glu Arg Gln Tyr

755 760 765

Lys Phe Leu Lys Ser Trp Ser Phe Phe Gly Lys Val Ser Gly Gln Val

770 775780

Ile Arg Ala Glu Lys Gly Ser Arg Phe Ala Ile Thr Leu Arg Glu His

785 790 795 800

Ile Asp His Ala Lys Glu Asp Arg Leu Lys Lys Leu Ala Asp Arg Ile

805 810 815

Ile Met Glu Ala Leu Gly Tyr Val Tyr Ala Leu Asp Glu Arg Gly Lys

820 825 830

Gly Lys Trp Val Ala Lys Tyr Pro Pro Cys Gln Leu Ile Leu Leu Glu

835 840 845

Glu Leu Ser Glu Tyr Gln Phe Asn Asn Asp Arg Pro Pro Ser Glu Asn

850 855 860

Asn Gln Leu Met Gln Trp Ser His Arg Gly Val Phe Gln Glu Leu Ile

865 870 875 880

Asn Gln Ala Gln Val His Asp Leu Leu Val Gly Thr Met Tyr Ala Ala

885 890 895

Phe Ser Ser Arg Phe Asp Ala Arg Thr Gly Ala Pro Gly Ile Arg Cys

900 905 910

Arg Arg Val Pro Ala Arg Cys Thr Gln Glu His Asn Pro Glu Pro Phe

915 920 925

Pro Trp Trp Leu Asn Lys Phe Val Val Glu His Thr Leu Asp Ala Cys

930 935940

Pro Leu Arg Ala Asp Asp Leu Ile Pro Thr Gly Glu Gly Glu Ile Phe

945 950 955 960

Val Ser Pro Phe Ser Ala Glu Glu Gly Asp Phe His Gln Ile His Ala

965 970 975

Asp Leu Asn Ala Ala Gln Asn Leu Gln Gln Arg Leu Trp Ser Asp Phe

980 985 990

Asp Ile Ser Gln Ile Arg Leu Arg Cys Asp Trp Gly Glu Val Asp Gly

995 1000 1005

Glu Leu Val Leu Ile Pro Arg Leu Thr Gly Lys Arg Thr Ala Asp Ser

1010 1015 1020

Tyr Ser Asn Lys Val Phe Tyr Thr Asn Thr Gly Val Thr Tyr Tyr Glu

1025 1030 1035 1040

Arg Glu Arg Gly Lys Lys Arg Arg Lys Val Phe Ala Gln Glu Lys Leu

1045 1050 1055

Ser Glu Glu Glu Ala Glu Leu Leu Val Glu Ala Asp Glu Ala Arg Glu

1060 1065 1070

Lys Ser Val Val Leu Met Arg Asp Pro Ser Gly Ile Ile Asn Arg Gly

1075 1080 1085

Asn Trp Thr Arg Gln Lys Glu Phe Trp Ser Met Val Asn Gln Arg Ile

1090 10951100

Glu Gly Tyr Leu Val Lys Gln Ile Arg Ser Arg Val Pro Leu Gln Asp

1105 1110 1115 1120

Ser Ala Cys Glu Asn Thr Gly Asp Ile

1125

Claims

1. A buffer system suitable for a Cas12 protein, the buffer system comprising a pH buffer; a divalent cation; a reducing agent; and a stabilizer;

the pH buffer is a pH buffer for maintaining the pH value at 7.0-9.0;

the divalent cations are magnesium ions;

the reducing agent is selected from DTT and/or beta-mercaptoethanol;

the stabilizer is selected from one or more of BSA, glycerol and PEG.

2. The buffer system of claim 1,

the final concentration of the pH buffer solution is 5-100 mM;

the final concentration of the divalent cation is 5-400 mM;

the final concentration of the reducing agent is 1-100 mM;

the final concentration of the stabilizer is 5-400 mug/ml.

3. The buffer system of claim 1 or 2, wherein the pH buffer is Tris buffer or HEPES buffer.

4. The buffer system of claim 1 or 2, wherein the divalent cation is magnesium ion; the reducing agent is selected from DTT; the stabilizer is selected from BSA.

5. Use of the buffer system according to any of claims 1 to 4, said use being any of the following:

I. use in increasing the in vitro activity of a Cas12 protein;

II. Use in improving efficiency of nucleic acid detection of a Cas12 protein;

and III, application in preparation of a nucleic acid detection kit based on the Cas12 protein.

6. A method of increasing an in vitro activity of a Cas12 protein, the method comprising placing the Cas12 protein in the buffer system of any one of claims 1-4 so as to exhibit the in vitro activity.

7. A method of nucleic acid detection using a Cas12 protein, the method comprising placing the Cas12 protein in the buffer system of any one of claims 1-4 to perform nucleic acid detection.

8. A method of using a Cas12 protein to bind and/or cleave nucleic acid in vitro, the method comprising providing a Cas12 protein in a buffer system of any one of claims 1-4 to bind and/or cleave nucleic acid in vitro.

9. A nucleic acid detection kit comprising a Cas12 protein and the buffer system of any one of claims 1-4 suitable for a Cas12 protein.