Method for producing single-stranded DNA using Casnicasse

Abstract

This invention discloses a method for producing single-stranded DNA using Cas nickase. This method solves the problem of limitations on the enzyme cleavage sites when using nicking endonucleases, and improves the nicking efficiency of plasmids by performing multiple nicking using multiple gRNAs in a single enzyme cleavage system, while also effectively improving the efficiency of subsequent digestion by exonucleases.

Description

Technical Field

[0001] [Cross - reference to Related Applications] This invention claims the priority of a previous application, Chinese Patent Application No. 2023107431619, with the invention title "Method for Producing Single - Stranded DNA Using Cas Nickase", filed with the China National Intellectual Property Administration on June 21, 2023. All the contents of this previous application are incorporated into this invention by reference.

[0002] [Technical Field] This invention belongs to the field of biochemistry. Specifically, it relates to a method for producing single - stranded DNA, particularly circular single - stranded DNA, related products, and kits.

Background Art

[0003] The CRISPR / Cas system plays a very important role in the field of modern bioscience, and its emergence has revolutionized genome editing technology. Gene knock - in based on the homology directed repair (HDR) pathway is one of its important applications. Due to template - dependence, the success and efficiency of gene knock - in are determined by an appropriate HDR template. Commonly used double - stranded DNA (dsDNA) templates have problems such as low efficiency, high off - target rate, and high cytotoxicity. Single - stranded DNA (ssDNA) has been demonstrated to be an ideal knock - in template for gene knock - in experiments based on HDR. As a knock - in template, ssDNA exhibits advantages such as excellent editing efficiency, low cytotoxicity, and low off - target effect. On the other hand, compared with linear single - stranded DNA (lssDNA), circular single - stranded DNA (cssDNA) has higher stability, can effectively and stably insert target fragments, has high target - directedness, and cssDNA donors are also superior to lssDNA donors in template - driven repair at target sites.

[0004] Currently, ssDNA synthesis methods are divided into chemical synthesis, bacterial-based synthesis, and enzymatic synthesis (see doi:10.3390 / genes11020116, US10940171B2). Chemical synthesis has limitations on ssDNA length, is costly, and requires additional purification steps. Phage-based synthesis allows for the mass production of ssDNA of any sequence in a bioreactor, but this method is time-consuming and unsuitable for rapid prototyping (see US20200362332). Enzymatic synthesis is a relatively low-cost and rapid method for synthesizing ssDNA, and it has fewer limitations on the length of the synthesized sequence, allowing for the effective synthesis of longer sequences.

[0005] One conventional enzymatic synthesis method for cssDNA involves first obtaining lssDNA and then cyclizing it with ligase to obtain cssDNA (see EP2610352B1 and CN107002292A). Another method involves using nicking endonucleases (e.g., Nb.BtsI, Nt.BspQI, Nb.Bpu10I) to nick the non-target strand of a double-stranded plasmid, digesting the cleaved DNA strand with an exonuclease, and finally obtaining the target cssDNA (see Zhang et al., Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA. Protein Expr Purif. 2010 Feb;69(2):226-34.doi:10.1016 / j.pep.2009.09.003;WO2023069948, WO1995009915A1).

[0006] While various methods exist for producing single-stranded DNA in this field, these methods have drawbacks such as high cost, long processing times, and the presence of residual dsDNA contamination. Therefore, there is still a need in this field for simple and easy-to-operate methods for producing high-purity single-stranded DNA. [Overview of the Initiative]

[0007] A first aspect of the present invention is: Step 1) prepares template DNA, which is a circular double-stranded DNA containing the target strand and the complementary strand, A step of adding gRNA and Cas nickase to template DNA, wherein the gRNA pairs complementaryally with a subregion of the complementary strand, and the Cas nickase is guided by the gRNA to form one or more nicks on the complementary strand (step 2), The present invention provides a method for producing circular single-stranded DNA, comprising step 3) digesting a complementary strand containing a nick with an exonuclease to obtain a digest product containing a target strand in the form of circular single-stranded DNA.

[0008] In one embodiment, the gRNA is sgRNA. In another specific embodiment, the Cas nickase is Cas9 nickase, for example, one or more Cas9 nickases selected from the group consisting of D10A, H840A, N863A, and N854A.

[0009] In one embodiment, the exonuclease is one or more selected from the group consisting of exonuclease T7, exonuclease ExoIII, and Lambda Exo.

[0010] In another embodiment, the template DNA is a plasmid. In a more specific embodiment, the plasmid includes an origin of replication and a target gene, but does not include a selection marker gene. The origin of replication is selected from, for example, the pUC origin of replication, the pMB1 and its derivative origins, the ColE1 origin of replication, and the R6Kγ origin of replication.

[0011] In one embodiment, the one or more nicknames are two or more nicknames.

[0012] In another embodiment, the ratio of the sequence length to the number of nicks of the template DNA is less than one nick per 10kb, preferably less than one nickeling site per 5kb, more preferably less than one nickeling site per 3kb, even more preferably less than one nickeling site per 2kb, and most preferably less than one nickeling site per 1kb.

[0013] In one embodiment, the molar ratio of Cas nicasse, gRNA, and template DNA is 3:3:1 to 12:12:1, preferably 4:4:1 to 11:11:1, and most preferably 5:5:1 to 10:10:1.

[0014] In one embodiment, the process further includes purifying the digested product after step 3) to obtain high-purity circular single-stranded DNA.

[0015] A second aspect of the present invention provides a method for producing linear single-stranded DNA, comprising the step of cleaving circular single-stranded (css) DNA obtained by the method described in the first aspect of the present invention to obtain linear single-stranded (lss) DNA.

[0016] A third aspect of the present invention provides a kit for producing circular or linear single-stranded DNA, comprising Cas niccas and exonuclease.

[0017] In one embodiment, the Cas nickase is Cas9 nickase, for example, one or more Cas9 nickases selected from the group consisting of D10A, H840A, N863A, and N854A.

[0018] In one embodiment, the exonuclease is one or more selected from the group consisting of exonuclease T7, exonuclease ExoIII, and Lambda Exo.

[0019] In another embodiment, the kit further comprises a positive reference template DNA and a positive gRNA. In yet another embodiment, the kit further comprises template DNA and gRNA. In yet another embodiment, the kit further comprises a buffer. In yet another embodiment, the kit further comprises reagents for purifying circular or linear single-stranded DNA.

[0020] A fourth aspect of the present invention provides circular or linear single-stranded DNA comprising a replication origin and a target gene, but not comprising a selection marker gene. In one embodiment, the replication origin is selected from pUC replication origin, pMB1 and its derivative replication origin, ColE1 replication origin, and R6Kγ replication origin.

[0021] A fifth aspect of the present invention provides a composition comprising cyclic or linear single-stranded DNA as described in the fourth aspect of the present invention. In one embodiment, the composition is a pharmaceutical composition.

[0022] A sixth aspect of the present invention provides a digest product containing a target strand in the form of a circular single-stranded DNA obtained by the method described in the first aspect of the present invention.

[0023] A seventh aspect of the present invention provides the use of a circular or linear single-stranded DNA as described in the fourth aspect of the present invention, or a composition as described in the fifth aspect of the present invention, or a digest product as described in the sixth aspect of the present invention, in gene therapy, genetic recombination, DNA library construction, DNA origami, or DNA memory elements. Beneficial technical effects

[0024] The DNA sequence widely contains a 5’-NGG-3’ sequence or other PAM sequences. According to the needs of experiments, multiple gRNA sequences can be designed to nick double-stranded DNA, which solves the problem that the enzyme cleavage site is limited when using nicking endonucleases conventionally. In addition, by performing multiple nicking using multiple gRNAs in one enzyme cleavage system, the nicking efficiency is improved, and the efficiency of subsequent digestion by exonuclease is effectively improved. Therefore, by producing circular single-stranded or linear single-stranded DNA by the method of the present invention, single-stranded DNA with high purity can be obtained easily, with simple operation and high efficiency.

Brief Description of Drawings

[0025] [Figure 1] Showing a closed circular single-stranded DNA (cssDNA) template plasmid (Fig. 1a shows the nicking site of Nt.BspQI, and Fig. 1b shows the nicking site of sgRNA). [Figure 2] Showing the enzyme cleavage products of Nt.BspQI nicking endonuclease. [Figure 3] Showing the enzyme cleavage products of Cas9_Nickase nicking endonuclease. [Figure 4] Showing the results of digesting the Nt.BspQI nick-introduced plasmid with exonuclease ExoIII. [Figure 5] Showing the results of digesting the Cas9_Nickase nick-introduced plasmid with exonuclease ExoIII. [Figure 6] Showing the nicking results of the template plasmid. [Figure 7] Showing the results of digestion with ExoIII for 2 hours. [Figure 8] Showing the results of digestion with ExoIII for 7 hours in the Nt.BspQI group. [Figure 9] Showing the comparison results of the purity of the closed circular single-stranded DNA (cssDNA) recovery product. [Figure 10]This shows the efficiency of producing closed circular single-stranded DNA (cssDNA) with different numbers of sgRNAs. [Modes for carrying out the invention]

[0026] Unless otherwise stated, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art. To facilitate understanding of the technical means provided herein, some technical terms are briefly explained below. technical terms

[0027] ssDNA Single-stranded DNA (ssDNA) lssDNA Linear single-stranded DNA (lssDNA) cssDNA Closed circular single-stranded DNA (cssDNA)

[0028] In this application, "template DNA" refers to circular double-stranded DNA, which is a starting material for producing single-stranded DNA. Similarly, "template plasmid" refers to a plasmid, which is a starting material for producing single-stranded DNA. The template DNA or template plasmid is a circular double-stranded DNA comprising a target strand and a complementary strand.

[0029] The term "circular" in "circular double-stranded DNA" and "circular single-stranded DNA" refers to DNA in which the above DNA is covalently bonded to form a ring.

[0030] The "target strand" and "complementary strand" of the template DNA refer to the DNA strand that is retained (i.e., the product) and the DNA strand that is nicked, respectively.

[0031] In the context of this application, the terms “nickases” and “nicking endonucleases” (also known as NEases or nickases) may be used interchangeably. Nickases recognize specific sites on double-stranded DNA, but unlike nucleases, nickases cleave one strand of the double-stranded DNA only at a specific site relative to the recognition site (doi:10.3390 / biom11101420). Taking the Cas9 protein as an example, the Cas9 nuclease cleaves two strands of a double-stranded DNA, while the Cas9 nickase is a variant of the Cas9 nuclease (mutated to inactivate one or more of its catalytic domains, for example, by introducing H840A into the HNH domain or D10A into the RuvC domain). Such variants retain the ability to bind to DNA based on gRNA specificity, but cleave only one strand of the double-stranded DNA (also called nicking), thus producing a single-stranded nick (DOI:10.1101 / gr.162339.113). For a description of various Cas nickases, see WO2023060256A1.

[0032] The inventors have found through their research that the current method of using a nicking endonuclease such as Nb.BtsI to nick the non-target strand of a double-stranded plasmid, digesting the cleaved DNA strand with an exonuclease, and finally obtaining the target cssDNA is theoretically simple and convenient, but has significant drawbacks in practical operation. For example, this method has very high demands on the enzyme cleavage sites. In order to subsequently nick and digest only the non-target strand of dsDNA, at least one target enzyme cleavage site must be present on the non-target strand (not all dsDNA fragments easily satisfy this condition), and all of these target enzyme cleavage sites must be present on the non-target strand and none on the target strand. Due to the above drawbacks, this method always requires modifying the dsDNA sequence to remove excess enzyme cleavage sites and retain only the enzyme cleavage sites at the target locations, which lengthens the production cycle. Due to the low nicking and digestion efficiency, a large amount of dsDNA remains in the final product, causing contamination and affecting downstream experiments.

[0033] To overcome the above-mentioned defects and other defects in the production of single-stranded RNA, the present invention uses a Cas nickase system (i.e., Cas nickase and gRNA combined therewith) to nick one strand of a circular double-stranded DNA template, thereby generating one or more nicks on that strand. Cas nickase is a variant form of Cas nuclease and can cleave only one strand of double-stranded DNA. Taking Cas9_D10A as an example, Cas9_D10A Nickase is a variant form of Cas9 nuclease and inactivates one active domain of Cas9 nuclease, so it can only recognize the PAM site by guide RNA and perform a single-strand break on single-stranded DNA complementary to the guide RNA. The PAM site is a short sequence (5'-NGG-3') located at the 3' end of the target DNA, and the guide RNA recognizes and binds to the complementary strand of this sequence, after which Cas9_D10A Nickase cleaves the recognized target DNA. Because PAM sites (e.g., 5'-NGG-3') are widely present in the DNA sequence, it is possible to select a 20nt or other appropriate length sequence as the gRNA sequence upstream of multiple sites with PAM sequences (e.g., GG sequences) at the 3' end, as needed. This solves the problem of limited enzyme cleavage sites when using nickeling end nucleases. Furthermore, performing multiple nickeling using multiple sgRNAs in a single enzyme cleavage system improves nickeling efficiency and effectively enhances the efficiency of subsequent digestion by exonucleases.

[0034] The present invention Step 1) involves preparing a template DNA which is a circular double-stranded DNA containing the target strand and the complementary strand (including, but not limited to, preparation, acquisition, and generation), A step of adding gRNA and Cas nickase to template DNA, wherein the gRNA pairs complementaryally with a subregion of the complementary strand, and the Cas nickase is guided by the gRNA to form one or more nicks on the complementary strand (step 2), The present invention provides a method for producing circular single-stranded DNA, comprising step 3) digesting a complementary strand containing a nick with an exonuclease to obtain a digest product containing a target strand in the form of circular single-stranded DNA.

[0035] Those skilled in the art will understand that the gRNA used in the present invention may be an sgRNA (single guide RNA), i.e., a single-molecule guide RNA, or a bimolecule guide RNA. Where gRNA is mentioned in this application, it is understood that sgRNA is also explicitly mentioned, to the extent that the context allows. In preferred embodiments, the gRNA is an sgRNA. Methods for designing preferred lengths, sequences, etc., of gRNAs containing sgRNA based on a template sequence are known in the art.

[0036] In the method of the present invention, it is preferable to design one or more, preferably multiple, gRNAs for a template DNA sequence, so that these gRNAs can find multiple nicking sites in the template DNA and generate multiple nicks. In a preferred embodiment, in step 2), multiple gRNAs, for example, 2 to 10 gRNAs, or 2 to 5 gRNAs, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 gRNAs, are added to the template DNA. The number of Cas nickases may also be one or more, for example, 1, 2, 3, or 4.

[0037] The Cas nickase used in the present invention may be any Cas protein having nickase function, that is, it is guided by gRNA to a specific target DNA sequence (i.e., complementary to gRNA) and performs nickase function to cleave only one strand of double-stranded DNA. In preferred embodiments, the Cas nickase is Cas9 nickase. The Cas9 nickase used in the present invention may be any Cas9 protein having nickase function, including, but not limited to, those in which one or more amino acid sites of D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and A987 have been mutated, including, but not limited to, Cas9 nickases such as D10A, H840A, N863A, and N854A. Multiple types of nickase may be used individually or in combination.

[0038] Non-exclusive examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also called Csn1 and Csx12), Cas10, Cas12, Cas13, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, and Csm Examples include 4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, or their functional variants (e.g., codon-optimized) or modifications. In some embodiments, Cas9 may be Cas9 derived from Streptococcus pyogenes or Streptococcus pneumoniae.

[0039] In some embodiments, the Cas nickase is Cas12 nickase (e.g., Cas12a nickase) or Cas13 nickase.

[0040] In this invention, any suitable exonuclease can be used to enzymatically digest the nicked DNA strand, which includes, but is not limited to, exonuclease T7, exonuclease ExoIII, and Lambda Exo. Due to their properties, exonucleases act only on or primarily on DNA with free 5' or 3' ends, and do not act on or have little effect on DNA with non-free 5' or 3' ends (e.g., covalently closed circular DNA). Therefore, exonucleases can digest and degrade the nicked DNA strand of the template DNA double helix, without affecting or significantly affecting the other non-nicked, covalently closed circular DNA strand.

[0041] In a preferred embodiment, the template DNA is a plasmid, i.e., plasmid double-stranded DNA. The plasmid may be a general plasmid and may include, for example, a backbone sequence, a promoter, an origin of replication, a selection marker gene (e.g., an antibiotic resistance gene or another selection marker gene), a multicloning site, a target gene sequence, and so on.

[0042] In a preferred embodiment, the template plasmid comprises an origin of replication and a target gene, but does not include a selection marker gene. The origin of replication is selected, for example, from the pUC origin of replication, the pMB1 and its derivative origins of replication, the ColE1 origin of replication, and the R6Kγ origin of replication. In another preferred embodiment, the plasmid comprises only the origin of replication and the target gene, but does not include a selection marker gene; that is, the plasmid consists of an origin of replication and a target gene, but does not include a selection marker gene. In yet another preferred embodiment, the plasmid consists substantially of an origin of replication and a target gene, but does not include a selection marker gene. For a method of producing the plasmid, refer to PCT / CN2021 / 133141, the entire application of which is cited by reference.

[0043] In one embodiment, the one or more nicks are two or more nicks, for example, 2 to 10 or 2 to 5 nicks, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nicks. In another embodiment, the ratio of the sequence length of the template DNA to the number of nicks is less than one nick per 10kb, preferably less than one nick per 5kb, more preferably less than one nick per 3kb, even more preferably less than one nick per 2kb, and most preferably less than one nick per 1kb.

[0044] In one embodiment, the molar ratio of Cas nickase such as Cas9 nickase to gRNA and template DNA is 3:3:1 to 12:12:1, preferably 4:4:1 to 11:11:1, and most preferably 5:5:1 to 10:10:1.

[0045] In a preferred embodiment, the process further includes purifying the digest product after step 3) to obtain high-purity circular single-stranded DNA. High purity means that the product is free of impurities, substantially free of impurities, or free of significant amounts of impurities. For example, calculated on a mass basis, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the DNA in the final product obtained by purification is circular single-stranded DNA. After digesting the complementary strand containing the nick using an exonuclease, impurities other than circular single-stranded DNA can be removed from the digest product by an appropriate purification method, which includes, but is not limited to, alcohol precipitation, column recovery, magnetic bead recovery, and phenol / chloroform / isoamyl alcohol recovery.

[0046] The present invention further provides a digest product comprising a target strand in the form of a circular single-stranded DNA obtained by the above method of the present invention. The digest product comprises circular single-stranded DNA, a product obtained by digesting a nickeled DNA strand with an exonuclease, gRNA, and Cas nickelase.

[0047] The present invention further provides a method for producing linear single-stranded DNA, comprising the step of cleaving the circular single-stranded (css) DNA obtained by the method of the present invention to obtain linear single-stranded (lss) DNA. In this step, any method known in the art for cleaving circular single-stranded DNA to obtain linear single-stranded DNA can be used. In a preferred embodiment, cleavage is performed at a specified site, for example, by using a Cas9 system for cleaving single-stranded DNA to perform site-directed cleavage.

[0048] The present invention further provides a kit for producing circular or linear single-stranded DNA, comprising Cas nickas and exonuclease. In one embodiment, the kit further comprises a positive reference template DNA and a positive gRNA for determining whether the components contained in the kit can produce circular or linear single-stranded DNA in a predetermined manner (e.g., a manner described in the instructions accompanying the kit). In another embodiment, the kit further comprises template DNA and gRNA. In yet another embodiment, the kit further comprises a buffer to facilitate the nicking reaction and / or exonuclease digestion reaction. In yet another embodiment, the kit further comprises a reagent for purifying the circular or linear single-stranded DNA.

[0049] The present invention further provides circular or linear single-stranded DNA comprising an origin of replication and a target gene, but not a selection marker gene. In one embodiment, the origin of replication is selected from pUC origin of replication, pMB1 and its derivative origins, ColE1 origin of replication, and R6Kγ origin of replication. In another preferred embodiment, the plasmid comprises only the origin of replication and the target gene, and does not include a selection marker gene; that is, the plasmid consists of an origin of replication and a target gene, and does not include a selection marker gene. In another preferred embodiment, the plasmid consists substantially of an origin of replication and a target gene, and does not include a selection marker gene. For a method of producing the plasmid, refer to PCT / CN2021 / 133141, the entire application of which is cited by reference.

[0050] The present invention further provides compositions comprising the above-mentioned cyclic or linear single-stranded DNA. In one embodiment, the composition is a pharmaceutical composition, which further comprises, but is not limited to, excipients commonly used in pharmaceuticals, such as fillers, disintegrants, lubricants, flavorings, and dyes.

[0051] The circular or linear single-stranded DNA, digest products containing circular single-stranded DNA, and compositions containing circular or linear single-stranded DNA according to the present invention can be suitably applied to gene therapy, genetic recombination (e.g., gene knock-in), construction of DNA libraries (e.g., DNA screening libraries), DNA origami, or DNA memory elements.

[0052] All references cited in this application are incorporated herein by reference in the same way that each reference is individually indicated as being incorporated herein by reference.

[0053] The technical means of the present invention will be described in more detail below with reference to the accompanying drawings, using examples. Unless otherwise stated, the methods and materials of the examples described below are all common products available on the market. Those skilled in the art will understand that the methods and materials described below are illustrative and should not be considered to limit the scope of the present invention in any way. The scope of the present invention is limited only by the claims. General experimental methods

[0054] (1) Construct a template plasmid for producing cssDNA, and design and synthesize a specific sgRNA based on the full-length sequence of the plasmid. The sgRNA guides the Cas9 Nickase nickeling endonuclease to perform site-specific nickeling at multiple sites on the template plasmid. As a control, the plasmid also includes three tandem-linked enzymatic cleavage sites of the nickeling endonuclease Nt.BspQI on the same DNA strand to improve the nickeling efficiency of Nt.BspQI.

[0055] (2) In the experimental group, the template plasmid was enzymatically cleaved with Cas9 Nickase to obtain open circular DNA with multiple cleavage sites on a single strand. In the control group, the template plasmid was enzymatically cleaved with Nt.BspQI to obtain open circular DNA with one cleavage site on a single strand. The open circular DNA derived from Cas9 Nickase and Nt.BspQI were digested with the exonuclease ExoIII to obtain target circular single-stranded DNA, and the purity of the two was compared. [Examples]

[0056] Example 1: Construction of a cssDNA template plasmid and design of sgRNA A template plasmid, pMF5-GFP-BspQI (Figure 1b), was constructed for the production of cssDNA. The full length of this plasmid is 3553 bp, and it was synthesized by the Genetics Division of Nanjing GenScript Biotechnology Co., Ltd. Its sequence can be viewed in the sequence listing. The full-length sequence of the pMF5-GFP-BspQI plasmid was imported into the sgRNA design website (http: / / crispor.tefor.net / ), and several sgRNA sequences were selected based on the required target sites and the full length of the plasmid. In this example, a total of five sgRNAs, each approximately 700 bp apart, were selected, and their sequences can be viewed in the sequence listing. They were synthesized by the Nucleic Acid Division of Nanjing GenScript Biotechnology Co., Ltd.

[0057] Furthermore, as the template plasmid for Nt.BspQI in the control group (Figure 1a), the plus-chain of pMF5-GFP-BspQI contains three closely linked Nt.BspQI enzyme cleavage sites to improve the nicking efficiency of the Nt.BspQI nicking endonuclease in the control group.

[0058] Example 2: Production of cssDNA using the nickel endonuclease Nt.BspQI / Cas9_Nickase 1. Production of Nick-Introduced Plasmids 1.1 Control Group: Production of Nick-Introduced Plasmids Using Nicking Endonuclease Nt.BspQI In the control group, the conventional nicking endonuclease Nt.BspQI was selected to produce nicking plasmids. The pMF5-GFP-BspQI plasmid was treated with Nt.BspQI to obtain open circular DNA with a single cleavage site per strand. The enzymatic cleavage system is shown in Table 1. Nt.BspQI was purchased from New England Biolabs, Inc., under product number R0644S.

[0059] [Table 1]

[0060] The enzyme cleavage reaction was carried out at 50°C and 500 rpm for 2.5 hours. A 1 μl sample was taken and verified by agarose gel electrophoresis. The enzyme cleavage results are shown in Figure 2, confirming that the enzyme cleavage reaction was complete. The sample was then thermally inactivated at 80°C for 20 minutes. A 20 μl sample was taken from the inactivated sample, stored in a refrigerator at 4°C, and used as a control for subsequent agarose gel electrophoresis.

[0061] Recovery of enzyme-cleaved samples using phenol / chloroform / isoamyl alcohol: 1) Equivolutes of phenol / chloroform / isoamyl alcohol (25:24:1) were added, vortexed for 10 seconds, and then centrifuged at 5000g for 5 minutes. 2) The upper aqueous phase was transferred to a new tube, and an equal volume of chloroform / isoamyl alcohol (24:1) was added. The mixture was vortexed for 10 seconds and centrifuged at 5000g for 5 minutes. 3) Repeat step 2). 4) The upper aqueous phase was transferred to a new tube. 1 / 10 the volume of 3M sodium acetate and 2.5 times the volume of ice-cold ethanol were added, mixed, and incubated at -20°C for 1 hour. 5) The mixture was centrifuged at 10,000 rpm for 10 minutes. 6) Discard the supernatant, gently wash the precipitate with 2000 μL of ice-cold 75% ethanol, and evaporate any remaining ethanol. 7) The plasmid was dissolved by adding an appropriate amount of DNase / RNase-free H2O.

[0062] 1.2 Experimental group: Production of nicked plasmids using the nicking endonuclease Cas9_Nickase To compare the nicking efficiency with that of conventional nicking endonucleases, Cas9_Nickase was selected in the experimental group to process the pMF5-GFP-BspQI plasmid, yielding open circular DNA with five cleavage sites per strand. The enzymatic cleavage system is shown in Table 2. Engenpy Cas9_Nickase was purchased from New England Biolabs, Inc., with product number M0650S.

[0063] [Table 2]

[0064] 5 μl of the sample was taken, 1 μl of proteinase K was added, and after standing at room temperature for 15 minutes, agarose gel electrophoresis was performed for verification. The enzyme cleavage results are shown in Figure 3, confirming that the enzyme cleavage reaction was complete. 100 μl of proteinase K was added to the reaction mixture, and after standing at room temperature for 15 minutes, it was thermally inactivated at 95°C for 10 minutes. 20 μl of the inactivated sample was taken and stored in a refrigerator at 4°C and used as a control for subsequent agarose gel electrophoresis.

[0065] As in Step 1.1, the enzyme-cleaved sample was recovered using phenol / chloroform / isoamyl alcohol.

[0066] 2. Production of cssDNA The nicked plasmids prepared in Steps 1.1 and 1.2 were digested with the exonuclease ExoIII, and the purity of the products obtained by digesting nicked plasmids from two different sources with ExoIII was compared. ExoIII was purchased from New England Biolabs, Inc., with product number M0206L.

[0067] 2.1 Digestion of Nt.BspQI-derived nicked plasmid with exonuclease ExoIII The nicked plasmid produced in Step 1.1 was digested with exonuclease ExoIII, and the digestion system is shown in Table 3.

[0068] [Table 3]

[0069] At 0.25 hours, 0.5 hours, 0.75 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 7 hours, and 24 hours, 20 μl of sample was taken from each sample. 11 mM EDTA was added to each sample, and after thermal inactivation at 70°C for 20 minutes, the samples were temporarily stored in a refrigerator at 4°C. After the digestion time was complete, the samples were used for verification by agarose gel electrophoresis.

[0070] The digestion results are shown in Figure 4. After 5 hours of digestion, the nick-introduced plasmid was still clearly present. After 24 hours of digestion, the nick-introduced plasmid was still not completely digested. The cssDNA content also gradually decreased with increasing digestion time. Agarose gel electrophoresis results were analyzed using the Tanon GIS series digital gel imaging system, and the specific ratios of cssDNA to remaining nick-introduced plasmid are shown in Table 4.

[0071] [Table 4]

[0072] 2.2 Digestion of Cas9 Nickase-derived Nick-introduced plasmids with exonuclease ExoIII The nicked plasmid produced in Step 1.2 was digested with exonuclease ExoIII, and the digestion system is shown in Table 5.

[0073] [Table 5]

[0074] At 0.25 hours, 0.5 hours, 0.75 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 7 hours, and 24 hours, 20 μl of sample was taken from each sample. 11 mM EDTA was added to each sample, and after thermal inactivation at 70°C for 20 minutes, the samples were temporarily stored in a refrigerator at 4°C and then used for verification by agarose gel electrophoresis.

[0075] The digestion results are shown in Figure 5. After 0.25 hours of digestion, a small amount of nick-introduced plasmid remained, but after 0.5 hours of complete digestion, no remaining nick-introduced plasmid was detected. After 24 hours of digestion, the cssDNA content decreased with increasing digestion time, but the band remained stable. Agarose gel electrophoresis results were analyzed using the Tanon GIS series digital gel imaging system, and the specific ratios of cssDNA to remaining nick-introduced plasmid are shown in Table 6.

[0076] [Table 6]

[0077] Comparing the conventional nickeling endonuclease Nt.BspQI with the Cas9_Nickase of the present invention, selecting the sgRNA-dependent nickeling endonuclease Cas9_Nickase and performing multiple nickeling on a template plasmid, followed by digesting the nickel-introduced plasmid with the exonuclease ExoIII, allows for the acquisition of higher purity cssDNA in a shorter time. Simultaneously, the digestion time by the exonuclease is significantly reduced, resulting in weaker degradation of the cssDNA product by the exonuclease, effectively improving the efficiency of cssDNA production.

[0078] Example 3: Comparison of recovery rates during cssDNA production between the Nt.BspQI group and the Cas9_Nickase group. In this example, the recovery rates of the Nt.BspQI group and the Cas9_Nickase group during cssDNA production were compared, using product purity as the recovery criterion.

[0079] 3.1 Production of Nick-Introduced Plasmids Nick-introduced plasmids were prepared using the nickel endonucleases Nt.BspQI and Cas9_Nickase, respectively. The initial dose of the template plasmid was 300 μg in both cases.

[0080] [Table 7]

[0081] The enzyme cleavage reaction was carried out at 50°C and 500 rpm for 3 hours. A 1 μl sample was taken and verified by agarose gel electrophoresis. The enzyme cleavage result is shown in lane 2 of Figure 6, confirming that the enzyme cleavage reaction was complete. The sample was thermally inactivated at 80°C for 20 minutes. A 20 μl sample was taken from the inactivated sample, stored in a refrigerator at 4°C, and used as a control for subsequent agarose gel electrophoresis.

[0082] [Table 8]

[0083] Three μl of the sample was taken, one μl of proteinase K was added, and the mixture was allowed to stand at room temperature for 15 minutes. Agarose gel electrophoresis was then performed to verify the enzyme cleavage result, which is shown in lane 3 of Figure 6, confirming that the enzyme cleavage reaction was complete. One hundred μl of proteinase K was added to the reaction mixture and allowed to stand at room temperature for 15 minutes, then it was thermally inactivated at 95°C for 10 minutes. Two hundred μl of the inactivated sample was taken and stored in a refrigerator at 4°C and used as a control for subsequent agarose gel electrophoresis.

[0084] 3.2 Recovery of Nick-Introduced Plasmid Recovery of enzyme-cleaved samples using phenol / chloroform / isoamyl alcohol: 1) Equivolutes of phenol / chloroform / isoamyl alcohol (25:24:1) were added, vortexed for 10 seconds, and then centrifuged at 5000g for 5 minutes. 2) The upper aqueous phase was transferred to a new tube, and an equal volume of chloroform / isoamyl alcohol (24:1) was added. The mixture was vortexed for 10 seconds and centrifuged at 5000g for 5 minutes. 3) Repeat step 2). 4) The upper aqueous phase was transferred to a new tube. 1 / 10 the volume of 3M sodium acetate and 2.5 times the volume of ice-cold ethanol were added, mixed, and incubated at -20°C for 1 hour. 5) The mixture was centrifuged at 10,000 rpm for 10 minutes. 6) Discard the supernatant, gently wash the precipitate with 2000 μL of ice-cold 75% ethanol, and evaporate any remaining ethanol. 7) 200 μl of DNase / RNase H2O solution was added to each group to dissolve the product. 8) One μl of sample was taken from each group, and the concentration was measured using NanoDrop® One Microvolume UV-Vis Spectrophotometers.

[0085] [Table 9]

[0086] 3.3 Production of cssDNA The recovered products from both groups in step 3.2 were digested with exonuclease ExoIII, and all recovered products were introduced into the digestive system. The digestive system is shown in Table 10. Finally, cssDNA was recovered using the purity of the product as the recovery criterion.

[0087] [Table 10]

[0088] After digestion at 37°C and 500 rpm for 2 hours, 1 μl samples were taken from each group and examined by agarose gel electrophoresis. The results are shown in Figure 7. In the Cas9_Nickase group, after 2 hours of digestion, the nickeld DNA band in lane 3 was converted to the high-purity cssDNA band in lane 5. However, in the Nt.BspQI group, after 2 hours of digestion, many nickeld DNA bands still remained and contaminated the cssDNA in lane 6. The product from the Cas9_Nickase group was temporarily stored in a refrigerator at 4°C.

[0089] The Nt.BspQI group was continuously digested, and samples were taken at 5, 6, and 7 hours. Agarose gel electrophoresis was performed to observe the digestion status, and the results are shown in Figure 8. After 7 hours of digestion, the remaining Nicked DNA was still not completely digested, and the content of the target product cssDNA also decreased with increasing digestion time.

[0090] The Nt.BspQI group was digested for up to 8 hours, and together with the product of the Cas9_Nickase group after 2 hours of digestion, it was recovered using phenol / chloroform / isoamyl alcohol. The recovery rates of the recovered products were compared, and agarose gel electrophoresis was performed again to verify the purity of the products. The recovery rates are shown in Table 11, and the agarose gel electrophoresis results are shown in Figure 9.

[0091] [Table 11]

[0092] As can be seen from the digestion results of the Nt.BspQI group, the exonuclease ExoIII not only digests and degrades nicked ssDNA, but also has a certain degree of degradation effect on circular ssDNA. When Nt.BspQI is selected as the nicking endonuclease to nick a single site of the template plasmid, the digested ssDNA fragment is too long, resulting in less-than-ideal digestion efficiency of the exonuclease ExoIII, low product purity, and severe contamination. Furthermore, attempting to increase product purity by extending the digestion time leads to degradation of the target product's cssDNA, reducing the final recovery efficiency.

[0093] Compared to the Nt.BspQI group, the Cas9_Nickase group significantly reduced the length of the digested fragments through nicking at multiple sites, improving the short-time digestion efficiency of the exonuclease ExoIII, effectively shortening the digestion time, improving the purity of the product, and resulting in a much higher recovery efficiency of the final product than the Nt.BspQI group.

[0094] Example 4: Design of sgRNA for cssDNA production using Cas9 Nickase Four groups of sgRNAs were designed using the plasmid sequence shown in Figure 1b (right) as a template. The lengths of the nickeling sites generated by Cas9_Nickase-sgRNA and the sequences between each nickeling site for each group are shown in Table 12. Nick-introduced DNA and cssDNA were produced for each of the four groups, and the production process was the same as in Example 2.

[0095] [Table 12]

[0096] The manufacturing results are shown in Figure 10. Lanes 2-5 show the manufacturing results for nicked DNA with sgRNA numbers of 5, 3, 2, and 1, respectively, while lanes 7-10 show the manufacturing results for cssDNA with sgRNA numbers of 5, 3, 2, and 1, respectively.

[0097] As shown in the results for lanes 9 and 10, when nicking with two or one sgRNA and digesting with ExoIII, the resulting product contains three forms of DNA, from top to bottom of the band: Product 1, which is the nicked-introduced DNA that was not completely digested by ExoIII; Product 2, which is the remaining parent plasmid DNA; and Product 3, which is the cssDNA of the target product, which accounts for the majority. The proportion of Product 1 and Product 2 in the group with one sgRNA (lane 10) was significantly higher than in the group with two sgRNAs (lane 9). As these results indicate, when designing sgRNAs according to the actual size of the template plasmid, the number of sgRNAs and the length of the break strands generated by nicking have a significant impact on the final production efficiency and purity of cssDNA, with the number of sgRNAs being two or more and the break strand length being less than 1.7kb.

[0098] The embodiments of the present invention are not limited to the above-described examples, and those skilled in the art can make various changes and improvements to the invention in form and detail without departing from the spirit and scope of the invention, all of which are deemed to fall within the scope of protection of the present invention. pMF5-GFP-BspQI (Sequence ID 1):

Claims

1. Step 1) prepares template DNA, which is a circular double-stranded DNA containing the target strand and the complementary strand, Step 2) involves adding gRNA and Cas nickase to template DNA, wherein the gRNA pairs complementaryally with a subregion of the complementary strand, and the Cas nickase is guided by the gRNA to form one or more nicks on the complementary strand. A method for producing circular single-stranded DNA, comprising step 3) digesting a complementary strand containing a nick with an exonuclease to obtain a digest product containing a target strand in the form of circular single-stranded DNA.

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

3. The method according to claim 1 or 2, wherein the Cas niccase is Cas9 niccase.

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

5. The method according to any one of claims 1 to 4, 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 claims 1 to 5, wherein the template DNA is a plasmid.

7. The method according to claim 6, wherein the plasmid comprises a replication origin and a target gene, but does not include a selection marker gene.

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

9. The method according to any one of claims 1 to 8, wherein the one or more nicks are two or more nicks.

10. The method according to any one of claims 1 to 9, wherein the ratio of the sequence length to the number of nicks of the template DNA is less than one nick per 10 kb, preferably less than one nickeling site per 5 kb, more preferably less than one nickeling site per 3 kb, even more preferably less than one nickeling site per 2 kb, and most preferably less than one nickeling site per 1 kb.

11. The method according to any one of claims 1 to 10, wherein the molar ratio of Cas nicasse, gRNA, and template DNA is 3:3:1 to 12:12:1, preferably 4:4:1 to 11:11:1, and most preferably 5:5:1 to 10:10:

1.

12. The method according to any one of claims 1 to 11, further comprising the step of purifying the digested product after step 3) to obtain high-purity circular single-stranded DNA.

13. A method for producing linear single-stranded DNA, comprising the step of cleaving a circular single-stranded (css) DNA obtained by the method of any one of claims 1 to 12 to obtain linear single-stranded (lss) DNA.

14. A kit for producing circular or linear single-stranded DNA, containing Cas niccas and exonuclease.

15. The kit according to claim 14, wherein the Cas nickase is Cas9 nickase.

16. The kit according to claim 15, wherein the Cas9 nickas 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 template DNA and gRNA.

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

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 molecule containing the origin of replication and target genes, but not containing a selection marker gene.

23. The cyclic or linear single-stranded DNA according to claim 22, wherein the replication origin is selected from a pUC replication origin, a pMB1 and its derivative replication origin, a ColE1 replication origin, and a R6Kγ replication origin.

24. A composition comprising the cyclic or linear single-stranded DNA described in claim 22 or 23.

25. The composition according to claim 24, which is a pharmaceutical composition.

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

27. Use of a circular or linear single-stranded DNA according to claim 22 or 23, or a composition according to claim 24 or 25, or a digest product according to claim 26, in gene therapy, genetic recombination, DNA library construction, DNA origami, or DNA memory elements.

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