Method for preparing single-stranded DNA by using Cas nickase

Abstract

The invention relates to a method for preparing single-stranded DNA (deoxyribonucleic acid) by using a Cas nickase, which solves the problem that the restriction enzyme cutting site is limited when the traditional nickase is used, and multiple gRNAs can be used in one restriction enzyme cutting system to carry out multi-point nickling, so that the digestion efficiency of subsequent exonuclease is effectively improved while the plasmid nickling efficiency is improved.

Description

A method for preparing single-stranded DNA using Cas nickase

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to a prior application, patent application number 2023107431619, filed with the State Intellectual Property Office of China on June 21, 2023, entitled “A Method for Preparing Single-Stranded DNA Using Cas Nickase.” The entire text of this prior application is incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of biochemistry, and specifically relates to a preparation method of single-stranded DNA, especially circular single-stranded DNA, related products and a kit. Background Art

[0004] The CRISPR / Cas system plays a crucial role in modern biological science, revolutionizing genome editing technology. Gene knock-in based on the homology-directed repair (HDR) pathway is one of its key applications. Due to its template-dependent nature, the success and efficiency of gene knock-in depend on the appropriate HDR template. Commonly used double-stranded DNA (dsDNA) templates suffer from low efficiency, high off-target rates, and significant cytotoxicity. Single-stranded DNA (ssDNA) has proven to be an ideal knock-in template for HDR-based gene knock-in experiments. As a knock-in template, ssDNA exhibits superior editing efficiency, minimal cytotoxicity, and reduced off-target effects. Compared to linear single-stranded DNA (lssDNA), circular single-stranded DNA (cssDNA) possesses greater stability, enabling efficient and stable insertion of target fragments with high on-target efficiency. cssDNA donors also outperform lssDNA donors in template-driven repair of target sites.

[0005] At present, the synthesis methods of ssDNA can be divided into chemical synthesis, bacterial-based synthesis and enzymatic synthesis (see: doi: 10.3390 / genes11020116; US10940171B2). Chemical synthesis has limitations on the length of ssDNA, is expensive, and requires additional purification methods. Phage-based synthesis can produce large quantities of ssDNA of any sequence in a bioreactor, but this method is time-consuming and not suitable for rapid prototyping (see: US20200362332). Enzymatic synthesis is a relatively low-cost and rapid method for ssDNA synthesis. This method has little restriction on the length of the synthesized sequence and can effectively synthesize longer sequences.

[0006] Regarding the traditional cssDNA enzymatic synthesis method, one method is to first obtain lssDNA and then circularize it with a ligase to obtain cssDNA (see: EP2610352B1; CN107002292A. Another method is to use a nicking enzyme (such as Nb.BtsI, Nt.BspQI, Nb.Bpu10I) to nick the non-target strand in the double-stranded plasmid, and then digest the broken DNA strand with a nuclease exonuclease to finally obtain the target cssDNA (Zhang et al., Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA. Protein Expr Purif. 2010Feb; 69(2): 226-34. doi: 10.1016 / j.pep.2009.09.003; WO 2023069948; WO1995009915A1).

[0007] Although there are many methods for preparing single-stranded DNA in the art, these methods have disadvantages such as high cost, long time consumption, or high dsDNA residual contamination. Therefore, there is still a need in the art for simple and easy-to-operate methods to prepare high-purity single-stranded DNA.

[0008] Summary of the Invention

[0009] The first aspect of the present invention provides a method for preparing circular single-stranded DNA, comprising the following steps:

[0010] 1) Providing a template DNA, wherein the template DNA is a circular double-stranded DNA comprising a target strand and a complementary strand;

[0011] 2) adding gRNA and Cas nickase to the template DNA, wherein the gRNA is complementary to a portion of the complementary strand, and the Cas nickase forms one or more nicks on the complementary strand under the guidance of the gRNA;

[0012] 3) Digesting the complementary strand containing the gap with an exonuclease to obtain a digestion product containing the target strand in the form of a circular single-stranded DNA.

[0013] In one embodiment, the gRNA is an sgRNA. In another specific embodiment, the Cas nickase is a Cas9 nickase, for example, the Cas9 nickase is one or more selected from the group consisting of D10A, H840A, N863A, and N854A.

[0014] In one embodiment, the exonuclease is one or more selected from the group consisting of Exonuclease T7, Exonuclease ExoIII, and Lambda Exo.

[0015] In another embodiment, the template DNA is a plasmid. In a more specific embodiment, the plasmid comprises a replication origin and a target gene, and does not comprise a screening tag gene. The replication origin is, for example, selected from the group consisting of a pUC replication origin, a pMB1 and its derivative replication origin, a ColE1 replication origin, and an R6Kγ replication origin.

[0016] In one embodiment, the one or more gaps are greater than or equal to two gaps.

[0017] In another embodiment, the ratio of the sequence length and the number of gaps of the template DNA is less than 1 gap per 10kb, preferably less than 1 nick site per 5kb, further preferably less than 1 nick site per 3kb, further preferably less than 1 nick site per 2kb, and most preferably less than 1 nick site per 1kb.

[0018] In one embodiment, the molar ratio of the Cas nickase to gRNA and template DNA is 3:3:1-12:12:1, preferably 4:4:1-11:11:1, and most preferably 5:5:1-10:10:1.

[0019] In one embodiment, after step 3), the method further comprises the following step: purifying the digestion product to obtain high-purity circular single-stranded DNA.

[0020] The second aspect of the present invention provides a method for preparing linear single-stranded DNA, comprising the following steps: cutting the circular single-stranded (css) DNA obtained by the method according to the first aspect of the present invention to obtain linear single-stranded (lss) DNA.

[0021] A third aspect of the present invention provides a kit for preparing circular or linear single-stranded DNA, comprising Cas nickase and exonuclease.

[0022] In one embodiment, the Cas nickase is a Cas9 nickase, for example, the Cas9 nickase is one or more selected from the group consisting of D10A, H840A, N863A, and N854A.

[0023] In one embodiment, the exonuclease is one or more selected from the group consisting of Exonuclease T7, Exonuclease ExoIII, and Lambda Exo.

[0024] In another embodiment, the kit further comprises a positive reference template DNA and a positive gRNA. In another embodiment, the kit further comprises a template DNA and a gRNA. In another embodiment, the kit further comprises a buffer. In another embodiment, the kit further comprises a reagent for purifying circular or linear single-stranded DNA.

[0025] A fourth aspect of the present invention provides a circular or linear single-stranded DNA comprising a replication initiation site and a target gene, and excluding a screening tag gene. In one embodiment, the replication initiation site is selected from the group consisting of a pUC replication initiation site, a pMB1 and its derivative replication initiation site, a ColE1 replication initiation site, and an R6Kγ replication initiation site.

[0026] The fifth aspect of the present invention provides a composition comprising the circular or linear single-stranded DNA according to the fourth aspect of the present invention. In one embodiment, the composition is a pharmaceutical composition.

[0027] The sixth aspect of the present invention provides a digestion product containing a target strand in the form of circular single-stranded DNA obtained according to the method of the first aspect of the present invention.

[0028] The seventh aspect of the present invention provides the use of the circular or linear single-stranded DNA as described in the fourth aspect of the present invention, or the composition as described in the fifth aspect of the present invention, or the digestion product as described in the sixth aspect of the present invention in gene therapy, gene recombination, DNA library construction, DNA origami or DNA storage elements.

[0029] Beneficial technical effects

[0030] In DNA sequences, 5'-NGG-3' or other PAM sequences are ubiquitous. According to experimental requirements, multiple gRNA sequences can be designed to nick double-stranded DNA, which solves the problem of limited enzyme cutting sites when using traditional nicking enzymes. In addition, multiple gRNAs are used in an enzyme cutting system to perform multi-point nicking, which improves the efficiency of nicking and effectively improves the digestion efficiency of subsequent nuclease exonucleases. Therefore, the method of the present invention is used to prepare circular single-stranded or linear single-stranded DNA, which is simple, easy to operate, efficient, and can obtain high-purity single-stranded DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1: Closed circular single-stranded DNA (cssDNA) template plasmid (Figure 1a: Nt.BspQI nicking site; Figure 1b: sgRNA nicking site)

[0032] Figure 2: Nt.BspQI nickase digestion products

[0033] Figure 3: Cas9_Nickase cleavage products

[0034] Figure 4: Results of digestion with exonuclease ExoIII for Nt.BspQI-nicked plasmid

[0035] Figure 5: Results of digestion of Cas9_Nickase nicking plasmid with exonuclease ExoIII

[0036] Figure 6: Template plasmid nicking results

[0037] Figure 7: ExoIII digestion results for 2h

[0038] Figure 8: Results of 7h digestion with ExoIII in the Nt.BspQI group

[0039] Figure 9: Comparison of the purity of recovered closed circular single-stranded DNA (cssDNA) products

[0040] Figure 10: Preparation efficiency of closed circular single-stranded DNA (cssDNA) with different numbers of sgRNAs DETAILED DESCRIPTION

[0041] Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. For the purpose of facilitating understanding of the technical solutions provided herein, some technical terms are briefly explained below.

[0042] Technical terms

[0043] ssDNA

[0044] single-stranded DNA (ssDNA)

[0045] lssDNA

[0046] Linear single-stranded DNA (lssDNA)

[0047] cssDNA

[0048] Closed circular single-stranded DNA (cssDNA)

[0049] In this application, "template DNA" refers to a circular double-stranded DNA that is used as the starting material for preparing single-stranded DNA. Similarly, "template plasmid" refers to a plasmid that is used as the starting material for preparing single-stranded DNA. The template DNA or template plasmid is a circular double-stranded DNA that contains a target strand and a complementary strand.

[0050] "Circular", such as "circular double-stranded DNA" and "circular single-stranded DNA", means that the DNA is covalently closed.

[0051] The "target strand" and "complementary strand" in the template DNA refer to the DNA strand intended to be retained (i.e., as the product) and the DNA strand to be nicked, respectively.

[0052] In the context of this application, the terms "nicking endonuclease" and "nicking enzyme" (also known as NEases or nickases) are used interchangeably. Nicking enzymes recognize specific sites in double-stranded DNA, but unlike nucleases, nicking enzymes only break one chain in the DNA double strand at a certain position relative to the recognition site (doi:10.3390 / biom11101420). Taking Cas9 protein as an example, Cas9 nuclease breaks two chains in the DNA double strand, while Cas9 nickase is a mutant of Cas9 nuclease (mutating one or more of the two or more catalytic domains to inactivate it, for example, by introducing H840A into the HNH domain or introducing D10A mutation into the Ruv C domain). This mutant retains the ability to bind DNA based on gRNA specificity, but is only able to cut (or nick) one chain in the DNA double strand, thereby generating a single-stranded gap (nick) (DOI:10.1101 / gr.162339.113). For a description of various Cas nickases, please see WO2023060256A1.

[0053] The inventors of the present invention have found through research that the current technology uses nicking enzymes such as Nb.BtsI to nick the non-target chain in the double-stranded plasmid, and then digests the broken DNA chain with a nuclease to finally obtain the target cssDNA. Although it is a simple and convenient method in theory, this method has significant defects in actual operation. For example, this method has very high requirements for enzyme cutting sites. In order to subsequently nick and digest only the non-target chain of dsDNA, it is necessary to have at least one target enzyme cutting site on the non-target chain (not all dsDNA fragments can easily meet this condition), and these target enzyme cutting sites are all located on the non-target chain and do not exist on the target chain at all. Due to the above shortcomings, this method often needs to modify the dsDNA sequence, remove redundant enzyme cutting sites, and retain only the enzyme cutting sites at the target position. This process prolongs the production cycle. Due to the low nicking efficiency and digestion efficiency, there is a high level of dsDNA residual contamination in the final product, which affects downstream experiments.

[0054] In order to overcome the above-mentioned and other defects in the production of single-stranded RNA, the present invention uses a Cas nickase system (i.e., a Cas nickase and a gRNA coordinated therewith) to nick one chain in a circular double-stranded DNA template, generating one or more gaps on the chain. Cas nickase is a mutant form of Cas nuclease that can only cut one chain in double-stranded DNA. Taking Cas9_D10A Cas nickase as an example, Cas9_D10A Nickase is a mutant form of Cas9 nuclease that inactivates an active domain of Cas9 nuclease, so it can only rely on guide RNA to recognize the PAM site and produce single-stranded cuts on single-stranded DNA complementary to the guide RNA. Wherein the PAM site is a short sequence (5'-NGG-3') located at the 3' end of the target DNA, the guide RNA recognizes and binds to the complementary chain of the sequence, and then Cas9_D10A Nickase shears the identified target DNA. PAM sites (such as 5'-NGG-3') are ubiquitous in DNA sequences. Therefore, according to needs, 20nt or other suitable lengths can be selected as gRNA sequences upstream of the 3' end containing a PAM sequence (such as a GG sequence) at multiple sites. This solves the problem of limited enzyme cutting sites when using traditional nicking enzymes. In addition, using multiple sgRNAs in one enzyme cutting system for multi-point nicking not only improves the nicking efficiency, but also effectively improves the subsequent exonuclease digestion efficiency.

[0055] The present invention provides a method for preparing circular single-stranded DNA, comprising the following steps:

[0056] 1) providing (including but not limited to providing, obtaining, generating) a template DNA, wherein the template DNA is a circular double-stranded DNA comprising a target strand and a complementary strand;

[0057] 2) adding gRNA and Cas nickase to the template DNA, wherein the gRNA is complementary to a portion of the complementary strand, and the Cas nickase forms one or more nicks on the complementary strand under the guidance of the gRNA;

[0058] 3) Digesting the complementary strand containing the gap with an exonuclease to obtain a digestion product containing the target strand in the form of a circular single-stranded DNA.

[0059] It will be understood by those skilled in the art that the gRNA used in the present invention may be sgRNA (single guide RNA), i.e., a guide RNA in the form of a single molecule, or a guide RNA in the form of a double molecule. When gRNA is mentioned in this application, it is understood that sgRNA is also explicitly mentioned where the context permits. In a preferred embodiment, the gRNA is sgRNA. Methods for designing gRNA including sgRNA based on template sequences, such as their preferred length, sequence, etc., are known in the art.

[0060] In the method of the present invention, one or more gRNAs are preferably designed for the template DNA sequence, preferably multiple, so that these gRNAs can find multiple nick sites on the template DNA and produce multiple cuts. In a preferred embodiment, multiple gRNAs, such as 2-10 gRNAs, or 2-5 gRNAs, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 gRNAs, are added to the template DNA in step 2). The number of Cas nickases can also be one or more, such as 1, 2, 3, or 4.

[0061] The Cas nickase used in the present invention can be any Cas protein with nickase function, that is, it can be guided by gRNA to a specific (i.e., complementary pairing with gRNA) target DNA sequence and can exercise the nickase function to cut and only cut one chain in the double-stranded DNA. In a preferred embodiment, the Cas nickase is a Cas9 nickase. The Cas9 nickase used in the present invention can be any Cas9 protein with nickase function, for example, including but not limited to mutation at one or more of the following amino acid sites: D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and A987, for example, including but not limited to the following Cas9 nickases: D10A, H840A, N863A and N854A. A variety of nickases can be used alone or in combination.

[0062] Non-limiting examples of Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12, Cas13, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Cs m3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, a homolog thereof, or a functional variant (e.g., codon-optimized) or modified form thereof. In some embodiments, Cas9 can be Cas9 from Streptococcus pyogenes or Streptococcus pneumoniae.

[0063] In some embodiments, the Cas nickase is a Cas12 nickase (e.g., a Cas12a nickase) or a Cas13 nickase.

[0064] In the present invention, any suitable exonuclease can be used to perform enzymatic digestion on the nicked DNA strand, including, for example, but not limited to, the following exonucleases: exonuclease T7, exonuclease ExoIII, and Lambda Exo. Due to the characteristics of exonucleases, they only or primarily act on DNA with free 5' or 3' ends, and do not or rarely act on DNA without free 5' or 3' ends (e.g., covalently closed circular DNA). Therefore, the exonuclease can digest and degrade the nicked DNA strand in the double-stranded template DNA without affecting or significantly affecting the other covalently closed circular DNA strand without a nick.

[0065] In a preferred embodiment, the template DNA is a plasmid, i.e., a double-stranded plasmid DNA. The plasmid can be a conventional plasmid, for example, comprising a backbone sequence, a promoter, a replication origin, a screening tag gene (e.g., an antibiotic resistance gene or other screening tag gene), a multiple cloning site, a target gene sequence, etc.

[0066] In a preferred embodiment, the template plasmid comprises a replication initiation site and a target gene, and does not comprise a screening tag gene. The replication initiation site is, for example, selected from the group consisting of a pUC replication initiation site, a pMB1 and its derivative replication initiation site, a ColE1 replication initiation site, and an R6Kγ replication initiation site. In another preferred embodiment, the plasmid only comprises a replication initiation site and a target gene, and does not comprise a screening tag gene, that is, the plasmid consists of a replication initiation site and a target gene, and does not comprise a screening tag gene. In another preferred embodiment, the plasmid consists essentially of a replication initiation site and a target gene, and does not comprise a screening tag gene. The preparation method of the plasmid can be found in PCT / CN2021 / 133141, which is cited in its entirety as a reference.

[0067] In one embodiment, the one or more gaps are greater than or equal to two gaps, such as 2-10 gaps, or 2-5 gaps, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 gaps. In another embodiment, the ratio of the sequence length of the template DNA to the number of gaps is less than 1 gap per 10 kb, preferably less than 1 gap per 5 kb, more preferably less than 1 gap per 3 kb, further preferably less than 1 gap per 2 kb, and most preferably less than 1 gap per 1 kb.

[0068] In one embodiment, the molar ratio of the Cas nickase, such as Cas9 nickase, gRNA and template DNA is 3:3:1-12:12:1, preferably 4:4:1-11:11:1, and most preferably 5:5:1-10:10:1.

[0069] In a preferred embodiment, after step 3), the following step is further included: purifying the digestion product to obtain high-purity circular single-stranded DNA. High purity means that it contains no impurities, or substantially no impurities, or does not contain a significant amount of impurities. For example, calculated by mass, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% of the total DNA in the final product obtained after purification is circular single-stranded DNA. After using nuclease to digest the complementary strand containing the gap, impurities other than the circular single-stranded DNA in the digestion product can be removed by a suitable purification method, including but not limited to, alcohol precipitation, column recovery, magnetic bead recovery, phenol / chloroform / isoamyl alcohol recovery, etc.

[0070] The present invention also provides a digestion product containing a target chain in the form of circular single-stranded DNA obtained by the above-mentioned method of the present invention. The digestion product contains circular single-stranded DNA, a product after exonuclease digestion of the nicked DNA chain, gRNA and Cas nickase.

[0071] The present invention also provides a method for preparing linear single-stranded DNA, comprising the steps of cleaving the circular single-stranded (css) DNA obtained by the method of the present invention to obtain linear single-stranded (lss) DNA. This step can use any method known in the art for cleaving circular single-stranded DNA to obtain linear single-stranded DNA. In a preferred embodiment, cleavage is performed at a selected site, for example, using a Cas9 system that cleaves single-stranded DNA for site-directed cleavage.

[0072] The present invention also provides a kit for preparing circular or linear single-stranded DNA, containing Cas nickase and exonuclease. In one embodiment, the kit also contains a positive reference template DNA and a positive gRNA, which are used to determine whether the components contained in the kit can produce circular or linear single-stranded DNA in a predetermined manner (e.g., the manner described in the instructions provided with the kit). In another embodiment, the kit also contains template DNA and gRNA. In another embodiment, the kit also contains a buffer to facilitate the nicking reaction and / or the exonuclease digestion reaction. In another embodiment, the kit also contains a reagent for purifying circular or linear single-stranded DNA.

[0073] The present invention also provides a circular or linear single-stranded DNA comprising a replication initiation site and a target gene, and does not comprise a screening tag gene. In one embodiment, the replication initiation site is selected from the group consisting of a pUC replication initiation site, a pMB1 and its derivative replication initiation site, a ColE1 replication initiation site, and an R6Kγ replication initiation site. In another preferred embodiment, the plasmid only comprises a replication initiation site and a target gene, and does not comprise a screening tag gene, that is, the plasmid consists of a replication initiation site and a target gene, and does not comprise a screening tag gene. In another preferred embodiment, the plasmid consists essentially of a replication initiation site and a target gene, and does not comprise a screening tag gene. The preparation method of the plasmid can be found in PCT / CN2021 / 133141, which is cited in its entirety as a reference.

[0074] The present invention also provides a composition comprising the circular or linear single-stranded DNA described above. In one embodiment, the composition is a pharmaceutical composition, which further comprises excipients commonly used in pharmaceutics, such as, but not limited to, fillers, disintegrants, lubricants, flavoring agents, pigments, and the like.

[0075] According to the present invention, the circular or linear single-stranded DNA, the digestion product containing the circular single-stranded DNA, and the composition containing the circular or linear single-stranded DNA can be suitably applied to gene therapy, gene recombination (such as gene knock-in), construction of DNA libraries (such as DNA screening libraries), DNA origami or DNA storage elements.

[0076] All references cited in this application are herein incorporated by reference to the same extent as if each individual reference were individually indicated to be incorporated by reference.

[0077] The technical solutions of the present invention will be further described in detail below by way of examples and in conjunction with the accompanying drawings. Unless otherwise stated, the methods and materials of the embodiments described below are all conventional products that can be purchased on the market. Those skilled in the art will understand that the methods and materials described below are merely exemplary and should not be construed as limiting the scope of the present invention in any way. The scope of the present invention is defined solely by the claims.

[0078] General experimental methods

[0079] (1) A template plasmid for preparing cssDNA was constructed, and specific sgRNA was designed and synthesized based on the full-length sequence of the plasmid. The sgRNA guided the Cas9_Nickase nicking enzyme to perform multiple site-specific nicking on the template plasmid. As a control, the plasmid also carried three tandem Nt.BspQI restriction sites located on the same DNA chain to improve the nicking efficiency of the nicking enzyme Nt.BspQI.

[0080] (2) In the experimental group, the template plasmid was digested with Cas9_Nickase to obtain open-circular DNA with multiple breaks on one strand; in the control group, the template plasmid was digested with Nt.BspQI to obtain open-circular DNA with one break on one strand. The open-circular DNA derived from Cas9_Nickase and Nt.BspQI was digested with exonuclease ExoIII to obtain the target circular single-stranded DNA, and the purity of the two was compared.

[0081] Example

[0082] Example 1: Construction of cssDNA template plasmid and design of sgRNA

[0083] The template plasmid pMF5-GFP-BspQI (Figure 1b) for preparing cssDNA was constructed. The plasmid is 3553 bp long and was synthesized by the Gene Department of Nanjing KingScript Biotechnology Co., Ltd. The sequence is shown in the sequence table. The full-length sequence of the pMF5-GFP-BspQI plasmid was imported into the sgRNA design website (http: / / crispor.tefor.net / ), and multiple sgRNA sequences were selected according to the desired target site and the full length of the plasmid. In this example, a total of 5 sgRNAs were selected, each separated by approximately 700 bp. The sequences are shown in the sequence table. Synthesized by the Nucleic Acid Department of Nanjing KingScript Biotechnology Co., Ltd.

[0084] At the same time, as the template plasmid of the control group Nt.BspQI (Figure 1a), the positive chain of pMF5-GFP-BspQI carries three tightly connected Nt.BspQI restriction sites to improve the nicking efficiency of the control group Nt.BspQI nicking enzyme.

[0085] Example 2: Preparation of cssDNA using Nt.BspQI / Cas9_Nickase

[0086] 1. Preparation of nicked plasmid

[0087] 1.1 Control group: Notch plasmid was prepared using notch enzyme Nt.BspQI.

[0088] In the control group, the traditional nickase Nt.BspQI was used to prepare the nicking plasmid. The pMF5-GFP-BspQI plasmid was treated with Nt.BspQI to obtain open-circular DNA with a single break in one strand. The enzyme digestion system is shown in Table 1. Nt.BspQI was purchased from New England Biotechnology Co., Ltd., Catalog No. R0644S.

[0089] Table 1 Nt.BspQI enzyme digestion system

[0090] Incubate the enzyme digestion at 50°C, 500 rpm for 2.5 hours. Take 1 µl of sample and run a gel to verify the digestion. The digestion results are shown in Figure 2, confirming that the digestion reaction is complete. Heat inactivate the sample at 80°C for 20 minutes. Take 20 µl of the inactivated sample and store it in a refrigerator at 4°C for use as a control in subsequent gel runs.

[0091] Phenol / chloroform / isoamyl alcohol recovery of enzyme-digested samples:

[0092] 1) Add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), vortex for 10 seconds, and then centrifuge at 5000g for 5 minutes;

[0093] 2) Transfer the upper aqueous phase to a fresh tube and add an equal volume of chloroform / isoamyl alcohol (24:1). Vortex for 10 seconds and centrifuge at 5000 g for 5 minutes.

[0094] 3) Repeat step 2;

[0095] 4) Transfer the upper aqueous phase to a new tube. Add 1 / 10 volume of 3M sodium acetate and 2.5 volumes of ice-cold ethanol, mix, and incubate at -20°C for 1 hour.

[0096] 5) Centrifuge at 10,000 rpm for 10 min;

[0097] 6) Pour off the supernatant and gently wash the precipitate with 2000 μL of 75% ice-cold ethanol, evaporating the remaining ethanol;

[0098] 7) Add appropriate amount of DNase / RNase-free H2O to dissolve the plasmid.

[0099] 1.2 Experimental group: Nick enzyme Cas9_Nickase was used to prepare the nick plasmid.

[0100] To compare the nicking efficiency of traditional nicking endonucleases, Cas9_Nickase was used to treat the pMF5-GFP-BspQI plasmid in this experimental group, generating open-circular DNA with five breaks on one strand. The enzyme digestion system is shown in Table 2. Engenpy Cas9_Nickase was purchased from New England Biotechnology Co., Ltd., Catalog No. M0650S.

[0101] Table 2 Cas9_Nickase enzyme digestion system

[0102] Add 1 μl of proteinase K to 5 μl of sample and incubate at room temperature for 15 minutes. Run the gel to verify the digestion. The digestion results are shown in Figure 3, confirming that the digestion reaction is complete. Add 100 μl of proteinase K to the total reaction, incubate at room temperature for 15 minutes, and then heat inactivate at 95°C for 10 minutes. Take 20 μl of the inactivated sample and store it in a refrigerator at 4°C for use as a control in subsequent gel runs.

[0103] The steps for recovering the enzyme-digested samples with phenol / chloroform / isoamyl alcohol are the same as in 1.1.

[0104] 2. Preparation of cssDNA

[0105] The nicked plasmids prepared in steps 1.1 and 1.2 were digested with exonuclease ExoIII to compare the purity of the products obtained from the two different sources after digestion. ExoIII was purchased from New England Biotechnology Co., Ltd., catalog number: M0206L.

[0106] 2.1 Digestion of Nt.BspQI-derived nicked plasmid with exonuclease ExoIII

[0107] Digest the nicked plasmid prepared in step 1.1 with exonuclease ExoIII. The digestion system is shown in Table 3.

[0108] Table 3 ExoIII digestion treatment of Nt.BspQI-derived nicked plasmid system

[0109] Take out 20ul of sample at 0.25h, 0.5h, 0.75h, 1h, 1.5h, 2h, 3h, 4h, 5h, 7h, and 24h respectively, add 11mM EDTA to each, heat inactivate at 70℃ for 20min, temporarily store in a 4℃ refrigerator, and use for gel running verification after the digestion time is completed.

[0110] The digestion results are shown in Figure 4. Significant residual nicked plasmid remained after 5 hours of digestion, and even after 24 hours, the nicked plasmid was still not completely digested. The cssDNA content also gradually decreased with increasing digestion time. Gel analysis using the Tanon GIS series digital gel imaging system reveals the specific ratio of cssDNA to residual nicked plasmid, as shown in Table 4.

[0111] Table 4 Ratio of cssDNA to residual nicked plasmid in Nt.BspQI nicked ExoIII digestion products

[0112] 2.2 Digestion of Cas9_Nickase-derived nicked plasmid with exonuclease ExoIII

[0113] The nicked plasmid prepared in step 1.2 was digested with exonuclease ExoIII. The digestion system is shown in Table 5.

[0114] Table 5 ExoIII digestion treatment of Cas9_Nickase-derived nicking plasmid system

[0115] Take out 20ul of sample at 0.25h, 0.5h, 0.75h, 1h, 1.5h, 2h, 3h, 4h, 5h, 7h, and 24h, respectively, add 11mM EDTA to each, heat inactivate at 70℃ for 20min, and temporarily store in a 4℃ refrigerator for subsequent gel running verification.

[0116] The digestion results are shown in Figure 5. A slight amount of residual nicked plasmid was observed after 0.25 hours of digestion, but complete digestion was achieved at 0.5 hours, with no detectable residual nicked plasmid. After 24 hours of digestion, the cssDNA content decreased with increasing digestion time, but the bands remained stable. Gel analysis using the Tanon GIS series digital gel imaging system reveals the ratio of cssDNA to residual nicked plasmid in Table 6.

[0117] Table 6 Ratio of cssDNA to residual nicked plasmid in Cas9_Nickase nicked ExoIII digestion products

[0118] Comparing the traditional nicking enzyme Nt.BspQI and the Cas9_Nickase of the present invention, the sgRNA-dependent nicking endonuclease Cas9_Nickase is selected to perform multi-point nicking on the template plasmid, and the nicked plasmid is digested with the nuclease ExoIII, which can obtain higher purity cssDNA in a shorter time. At the same time, since the exonuclease digestion time is greatly shortened, the exonuclease also has little degradation effect on the product cssDNA, effectively improving the efficiency of cssDNA preparation.

[0119] Example 3: Comparison of recovery rates of cssDNA prepared by Nt.BspQI group / Cas9_Nickase group

[0120] In this example, the recovery rates of cssDNA prepared by the Nt.BspQI group / Cas9_Nickase group were compared using product purity as the recovery standard.

[0121] 3.1 Preparation of nicked plasmid

[0122] Nicking plasmids were prepared using Nt.BspQI and Cas9_Nickase, respectively. The initial input of template plasmids was 300 μg.

[0123] Table 7 Nt.BspQI enzyme digestion system

[0124] Incubate the enzyme digestion reaction at 50°C, 500 rpm for 3 hours. Take 1 μl of sample and run a gel to verify the digestion. The digestion result is shown in lane 2 of Figure 6, confirming that the digestion reaction is complete. Heat inactivate the reaction at 80°C for 20 minutes. Take 20 μl of the inactivated sample and store it in a refrigerator at 4°C for use as a control in subsequent gel runs.

[0125] Table 8 Cas9_Nickase enzyme digestion system

[0126] Add 1 μl of proteinase K to 3 μl of sample, incubate at room temperature for 15 minutes, and run a gel to verify the digestion. The digestion result is shown in lane 3 of Figure 6, confirming that the digestion reaction is complete. Add 100 μl of proteinase K to the total reaction, incubate at room temperature for 15 minutes, and then heat inactivate at 95°C for 10 minutes. Take 20 μl of the inactivated sample and store it in a refrigerator at 4°C for use as a control in subsequent gel runs.

[0127] 3.2 Recovery of nicked plasmids

[0128] Phenol / chloroform / isoamyl alcohol recovery of enzyme-digested samples:

[0129] 1) Add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), vortex for 10 seconds, and then centrifuge at 5000g for 5 minutes;

[0130] 2) Transfer the upper aqueous phase to a fresh tube and add an equal volume of chloroform / isoamyl alcohol (24:1). Vortex for 10 seconds and centrifuge at 5000 g for 5 minutes.

[0131] 3) Repeat step 2;

[0132] 4) Transfer the upper aqueous phase to a new tube. Add 1 / 10 volume of 3M sodium acetate and 2.5 volumes of ice-cold ethanol, mix, and incubate at -20°C for 1 hour.

[0133] 5) Centrifuge at 10,000 rpm for 10 min;

[0134] 6) Pour off the supernatant and gently wash the precipitate with 2000 μL of 75% ice-cold ethanol, evaporating the remaining ethanol;

[0135] 7) Add 200ul of DNase / RNase H2O solution to each group;

[0136] 8) Take 1ul sample from each group and use NanoDrop TM One Microvolume UV-Vis Spectrophotometers. Determine concentration.

[0137] Table 9 Notched plasmid recovery efficiency

[0138] 3.3 cssDNA preparation

[0139] Digest the two groups of recovered products from step 3.2 separately with exonuclease ExoIII. All recovered products are then added to the digestion system. The digestion system is shown in Table 10. Finally, cssDNA is recovered using product purity as the recovery criterion.

[0140] Table 10 ExoIII digestion system

[0141] After 2 hours of digestion at 37°C and 500 rpm, 1 μl of sample from each group was run on an agarose gel for verification. The results are shown in Figure 7. In the Cas9_Nickase group, after 2 hours of digestion, the nicked DNA band in lane 3 transformed into a highly pure cssDNA band in lane 5. However, after 2 hours of digestion in the Nt.BspQI group, the css DNA in lane 6 still contained significant contamination with the nicked DNA band. The product from the Cas9_Nickase group was temporarily stored in a 4°C refrigerator.

[0142] Digestion continued in the Nt.BspQI group, with samples taken at 5, 6, and 7 hours, and digestion progress observed by gel running, as shown in Figure 8. At 7 hours of digestion, residual nicked DNA was still not completely digested, and the content of the target product, cssDNA, decreased with prolonged digestion.

[0143] Digestion was continued for 8 hours in the Nt.BspQI group and the product from the 2-hour digestion in the Cas9_Nickase group, and then recovered using phenol / chloroform / isoamyl alcohol. The recovered products were compared for recovery rate and then re-run on an agarose gel to verify product purity. The recovery rates are shown in Table 11, and the results are shown in Figure 9.

[0144] Table 11 Comparison of cssDNA recovery efficiency

[0145] The digestion results of the Nt.BspQI group show that in addition to digesting and degrading nicked ssDNA, the exonuclease ExoIII also has a certain degradation effect on circular ssDNA. When Nt.BspQI is selected as the nicking enzyme to nick a single site on the template plasmid, the length of the ssDNA fragment to be digested is too long, and the digestion efficiency of the exonuclease ExoIII is not ideal, resulting in low product purity and heavy contamination. Attempting to increase product purity by extending the digestion time will lead to degradation of the target product cssDNA, resulting in a reduction in the final recovery efficiency.

[0146] Compared with the Nt.BspQI group, the Cas9_Nickase group shortened the length of the fragment to be digested to a great extent through multi-site nicking, improved the digestion efficiency of the nuclease ExoIII in a short period of time, effectively reduced the digestion time while improving the product purity, and the recovery efficiency of the final product was also much higher than that of the Nt.BspQI group.

[0147] Example 4: Design of sgRNA for Cas9_Nickase Preparation of cssDNA

[0148] Using the plasmid sequence in Figure 1b (right) as a template, four groups of different sgRNAs were designed. The nick sites generated by the Cas9_Nickase-sgRNA in each group and the length of the sequence between each nick site are shown in Table 12. Nicked DNA and cssDNA were prepared in each of the four groups, following the same preparation process as in Example 2.

[0149] Table 12 Number of nick sites and length of each segment

[0150] The preparation results are shown in Figure 10. Lanes 2-5 show the nicked DNA preparation results when the number of sgRNAs is 5, 3, 2, and 1, respectively, and lanes 7-10 show the cssDNA preparation results when the number of sgRNAs is 5, 3, 2, and 1, respectively.

[0151] As shown in the results of lanes 9 and 10, when two or one sgRNAs are selected for nicking and ExoIII digestion, three forms of DNA are obtained in the product. The bands include, from top to bottom, product 1: nicked DNA that is not completely digested by ExoIII, product 2: residual parental plasmid DNA, and product 3: target product cssDNA that accounts for the majority. Among them, the proportion of products 1 and 2 in the one sgRNA group (lane 10) is significantly higher than that in the two sgRNA group (lane 9). This result shows that when designing sgRNA according to the actual size of the template plasmid, the number of sgRNAs and the length of the broken chain caused by the nick have a crucial influence on the preparation efficiency and purity of the final cssDNA. The number of sgRNAs should be no less than 2, and the length of the broken chain should be less than 1.7kb.

[0152] The embodiments of the present invention are not limited to the above embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and details, and these are all considered to fall within the scope of protection of the present invention.

[0153] pMF5-GFP-BspQI (SEQ ID NO:1):

Claims

1. A method for preparing circular single-stranded DNA, comprising the following steps: 1) Providing a template DNA, wherein the template DNA is a circular double-stranded DNA comprising a target strand and a complementary strand; 2) adding gRNA and Cas nickase to the template DNA, wherein the gRNA and a partial region of the complementary chain are complementary and paired, wherein the Cas nickase forms one or more gaps on the complementary chain under the guidance of the gRNA; 3) Digest the complementary strand containing the gap with an exonuclease to obtain a digestion product containing the target strand in the form of a circular single-stranded DNA.

2. The method of claim 1, wherein the gRNA is sgRNA.

3. The method of claim 1 or 2, wherein the Cas nickase is a Cas9 nickase.

4. The method of claim 3, wherein the Cas9 nickase is one or more selected from the group consisting of D10A, H840A, N863A, and N854A.

5. The method according to any one of the preceding claims, wherein the exonuclease is one or more selected from the group consisting of Exonuclease T7, Exonuclease ExoIII, and Lambda Exo.

6. The method according to any one of the preceding claims, wherein the template DNA is a plasmid.

7. The method of claim 6, wherein the plasmid comprises a replication origin and a target gene, and does not comprise a screening tag gene.

8. The method according to claim 7, wherein the replication origin site is selected from the group consisting of a pUC replication origin site, a pMB1 and its derivative replication origin site, a ColE1 replication origin site and a R6Kγ replication origin site.

9. The method of any preceding claim, wherein the one or more gaps are greater than or equal to two gaps.

10. The method according to any of the preceding claims, wherein the ratio of the sequence length of the template DNA to the number of gaps is less than 1 gap per 10 kb, preferably less than 1 nick site per 5 kb, further preferably less than 1 nick site per 3 kb, further preferably less than 1 nick site per 2 kb, and most preferably less than 1 nick site per 1 kb.

11. A method as described in any one of the above claims, wherein the molar ratio of the Cas nickase to the gRNA and the template DNA is 3:3:1-12:12:1, preferably 4:4:1-11:11:1, and most preferably 5:5:1-10:10:

1.

12. The method according to any one of the preceding claims, further comprising the following step after step 3): purifying the digestion product to obtain a highly pure circular single-stranded DNA.

13. A method for preparing linear single-stranded DNA, comprising the step of cleaving the circular single-stranded (css) DNA obtained according to the method of any one of the preceding claims to obtain linear single-stranded (lss) DNA.

14. A kit for preparing circular or linear single-stranded DNA, comprising Cas nickase and exonuclease.

15. The kit of claim 14, wherein the Cas nickase is a Cas9 nickase.

16. The kit of claim 15, wherein the Cas9 nickase is one or more selected from the group consisting of D10A, H840A, N863A, and N854A.

17. The kit according to any one of claims 14 to 16, wherein the exonuclease is one or more selected from the group consisting of exonuclease T7, exonuclease ExoIII, and Lambda Exo.

18. The kit according to any one of claims 14 to 17, further comprising a positive reference template DNA and a positive gRNA.

19. The kit according to any one of claims 14 to 18, further comprising a template DNA and a gRNA.

20. The kit according to any one of claims 14 to 19, further comprising a buffer.

21. The kit according to any one of claims 14 to 20, further comprising a reagent for purifying circular or linear single-stranded DNA.

22. A circular or linear single-stranded DNA comprising a replication origin and a target gene, and not comprising a selection tag gene.

23. The circular or linear single-stranded DNA according to claim 22, wherein the replication initiation site is selected from the group consisting of a pUC replication initiation site, a pMB1 and its derivative replication initiation site, a ColE1 replication initiation site and a R6Kγ replication initiation site.

24. A composition comprising the circular or linear single-stranded DNA according to claim 22 or 23.

25. The composition of claim 24, which is a pharmaceutical composition.

26. A digestion product containing a target strand in the form of circular single-stranded DNA obtained according to the method of any one of claims 1 to 11.

27. Use of the circular or linear single-stranded DNA according to claim 22 or 23, or the composition according to claim 24 or 25, or the digestion product according to claim 26 in gene therapy, gene recombination, DNA library construction, DNA origami or DNA storage element.

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