Circular single-stranded DNA molecule for recombinase-based gene editing

Abstract

Circular single-stranded DNA (cssDNA) molecules suitable for recombinase-based gene editing are described herein. Double-stranded DNA (dsDNA) molecules, kits containing dsDNA molecules for preparing cssDNA molecules, and kits and methods for performing genome editing using cssDNA or dsDNA are also described herein.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 457,420, filed on April 6, 2023, the entire disclosure of which is hereby incorporated by reference herein.

[0002] Sequence Listing This application includes a sequence listing submitted in XML (ST.26) format, the entire disclosure of which is hereby incorporated by reference herein. The name of the XML copy created on April 4, 2024 is "385722 - 1003WO1_Seq_Listing.xml" and the size is 13,523 bytes.

Background Art

[0003] Background Recombinase - based gene editing, such as integrase - based gene editing, requires delivery of a high concentration of double - stranded DNA donor template into host cells to achieve efficient integration. However, the presence of double - stranded DNA in host cells causes cytotoxicity, which reduces the integration efficiency. Due to the dilemma between the need for a high donor template concentration and the resulting cytotoxicity, the editing efficiency achievable with the current version of this technology is significantly limited.

[0004] There is a need for a new strategy for recombinase - based gene editing that can achieve high integration efficiency and low cytotoxicity. The present invention addresses this need.

Summary of the Invention

[0005] Summary In some aspects, the present invention is directed to the following non - limiting embodiments.

[0006] Circular single - stranded DNA In several aspects, the present invention is directed toward circular single-stranded DNA (cssDNA) molecules.

[0007] In some embodiments, the cssDNA molecule contains a recombinase-recognition sequence that is recognized by a recombinase.

[0008] In some aspects, recombinases are site-specific recombinases (SSRs).

[0009] In some embodiments, site-specific recombinases are tyrosine integrases, serine integrases, or site-specific transposases.

[0010] In some embodiments, the recombinase recognition sequence is an X-over P1 locus (loxP) sequence, a flippase recognition target (FRT) sequence, a rox sequence, an attachment-phage (attP) sequence, an attachment-bacteria (attB) sequence, an attachment-left (attL) sequence, an attachment-right (attR) sequence, a VloxP sequence, a vox sequence, or a transposase recognition inverted terminal repeat (ITR) sequence.

[0011] In some embodiments, the cssDNA molecule further includes a payload sequence.

[0012] In some embodiments, the payload sequence encodes an RNA molecule or a polypeptide.

[0013] In some embodiments, the payload sequence contains a length of approximately 1 nucleotide to approximately 50,000 nucleotides.

[0014] In some embodiments, the cssDNA molecule is purified or concentrated.

[0015] double-stranded DNA In several aspects, the present invention is directed towards double-stranded DNA (dsDNA) molecules.

[0016] In some embodiments, the dsDNA comprises a template sequence encoding a cssDNA containing a recombinase-recognition sequence recognized by a recombinase, and a cssDNA conversion sequence.

[0017] In some embodiments, the double-stranded DNA molecule is a linear dsDNA molecule or a circular dsDNA molecule.

[0018] In some embodiments, the cssDNA conversion sequence is a sequence that originates from or is derived from the M13 phage f1 origin, M13 phage replication initiator, M13 phage replication terminator, or M13 phage packing signal (PS).

[0019] In some embodiments, the dsDNA further comprises a payload sequence, or a payload sequence insertion site for inserting a payload sequence.

[0020] In some embodiments, the payload insertion site is a multiple cloning site.

[0021] In some embodiments, ds DNA is a single DNA molecule or two or more distinct DNA molecules.

[0022] kit In several aspects, the present invention is directed toward a kit.

[0023] In some embodiments, the kit comprises a cssDNA molecule, or a dsDNA molecule and a system for converting the dsDNA molecule to cssDNA, and a recombinase or a nucleic acid encoding a recombinase.

[0024] In some embodiments, the cssDNA molecule or dsDNA molecule is identical or similar to those described herein.

[0025] In some embodiments, the recombinase recognizes the recombinase recognition sequence of cssDNA.

[0026] In some embodiments, the recombinase is a serine integrase, a tyrosine integrase, or a site-specific transposase.

[0027] In some embodiments, the recombinase is Cre, Flp, Dre, SCre, VCre, Vika, B2, B3, KD, ΦC31, Bxb1, RDF, λ, HK022, HP1, γδ, ParA, Gin, R4, TP901-1, TG1, PhiRv1, PhiBT1, SprA, XisF, TnpX, R, A118, spoIVCA, MR11, SCCmec, TndX, XerC, XerD, XisA, Hin, Cin, mrpA, beta, PhiFC1, Fre, Clp, sTre, FimE, HbiF, φFC1, φC1, q 370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, R2, the retrotransposase encoded by L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1), Minos, R1, R2, R3, R4, R5, Mu transposase, Tn3 transposase, Tn5 transposase, Tn7 transposase, Tol2, Sleeping Beauty transposase, or PiggyBac transposase.

[0028] In some embodiments, the kit includes a nucleic acid encoding a recombinase, which may be mRNA, single-stranded DNA, or double-stranded DNA.

[0029] In some embodiments, the kit further comprises oligonucleotides that are fully or partially complementary to the cssDNA molecule.

[0030] In some embodiments, oligonucleotides are primers that convert cssDNA molecules into circular double-stranded DNA molecules within cells.

[0031] In some embodiments, the oligonucleotides have a length of approximately 5 to 200 nucleotides.

[0032] In some embodiments, the kit contains a nucleic acid encoding a recombinase, and the nucleic acid encoding the recombinase is part of a cssDNA molecule or a dsDNA molecule.

[0033] In some embodiments, the kit further comprises cssDNA and components for introducing a recombinase or nucleic acid encoding a recombinase into a cell.

[0034] system In several aspects, the present invention is directed towards systems.

[0035] In some embodiments, the system includes the kit and the cells to be manipulated according to this specification.

[0036] In some embodiments, the genomic DNA or mitochondrial DNA of a cell includes a landing pad sequence for the incorporation of cssDNA molecules into the genomic DNA or mitochondrial DNA.

[0037] In some embodiments, the landing pad sequence is recognized by a recombinase, which then cleaves the cssDNA molecule and inserts the linearized cssDNA molecule into genomic DNA or mitochondrial DNA using the landing pad sequence as an anchor site.

[0038] In some embodiments, the landing pad sequence is an X-over P1 locus (loxP) sequence, a flippase recognition target (FRT) sequence, a rox sequence, an attachment phage (attP) sequence, an attachment bacterium (attB) sequence, an attachment left (attL) sequence, an attachment right (attR) sequence, a VloxP sequence, a vox sequence, or a transposase recognition inverted terminal repeat (ITR) sequence.

[0039] In some embodiments, the cells are bacterial cells, plant cells, or mammalian cells.

[0040] In some embodiments, the cells are isolated cells, cells derived from a cell line, primary cells, or cells under study.

[0041] In some embodiments, the cells are pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), B cells, T cells, natural killer (NK) cells, or terminally differentiated non-dividing cells such as neurons, muscle cells, or hepatocytes.

[0042] Methods for performing genetic manipulation In some aspects, the present invention is directed toward a method for performing genetic manipulation in cells.

[0043] In some embodiments, the method includes the step of introducing cssDNA as defined herein and a recombinase or nucleic acid encoding a recombinase into a cell.

[0044] In some embodiments, the recombinase recognizes a recombinase-recognition sequence of the cssDNA for incorporating the cssDNA into the cell's genomic DNA or mitochondrial DNA.

[0045] In some embodiments, the recombinase is a serine recombinase, a tyrosine recombinase, or a site-specific transposase.

[0046] In some embodiments, the recombinase is Cre, Flp, Dre, SCre, VCre, Vika, B2, B3, KD, ΦC31, Bxb1, RDF, λ, HK022, HP1, γδ, ParA, Gin, R4, TP901-1, TG1, PhiRv1, PhiBT 1, SprA, XisF, TnpX, R, A118, spoIVCA, MR11, SCCmec, TndX, XerC, XerD, XisA, Hin, Cin, mrpA, beta, PhiFC1, Fre, Clp, sTre, FimE, HbiF, φFC1, φC1, q Retrotransposases coded by 370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, R2, L1, Tol2 Tc1, Tc3, Mariner(Himar 1), Mariner(mos 1) Minos, R1, R2, R3, R4, R5, Mu transposase, Tn3 transposase, Tn5 transposase, Tn7 transposase, Tol2, Sleeping Beauty transposase, or PiggyBac transposase.

[0047] In some embodiments, the method further includes the step of introducing into cells an oligonucleotide that is fully or partially complementary to at least a portion of the cssDNA molecule.

[0048] In some embodiments, the cellular mechanisms of a cell convert cssDNA molecules into circular double-stranded DNA molecules using oligonucleotides as primers.

[0049] In some embodiments, the oligonucleotides have a length of approximately 5 to 200 nucleotides.

[0050] In some embodiments, the method includes the step of introducing sense strand cssDNA, antisense strand cssDNA, or a mixture of both into cells.

[0051] In some embodiments, the cellular genomic DNA or mitochondrial DNA includes a landing pad sequence for the integration of cssDNA molecules into the genomic DNA or mitochondrial DNA.

[0052] In some embodiments, the method further includes the step of culturing cells under conditions sufficient to allow a landing pad sequence to be recognized by a recombinase, and sufficient for the recombinase to cleave a cssDNA molecule and insert the linearized cssDNA molecule into genomic DNA or mitochondrial DNA using the landing pad sequence as an anchor site.

[0053] In some embodiments, the landing pad sequence is an X-over P1 locus (loxP) sequence, a flippase recognition target (FRT) sequence, a rox sequence, an attachment phage (attP) sequence, an attachment bacterium (attB) sequence, an attachment left (attL) sequence, an attachment right (attR) sequence, a VloxP sequence, a vox sequence, or a transposase recognition inverted terminal repeat (ITR) sequence.

[0054] In some embodiments, the cells are bacterial cells, plant cells, or mammalian cells.

[0055] In some embodiments, the cells are isolated cells, cells derived from a cell line, primary cells, or cells under study.

[0056] In some embodiments, the cells are pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), B cells, T cells, natural killer (NK) cells, or terminally differentiated non-dividing cells such as neurons, muscle cells, or hepatocytes. [Brief explanation of the drawing]

[0057] The following detailed description of exemplary embodiments will be better understood when read in conjunction with the accompanying drawings. Non-limiting embodiments are shown in the drawings for illustrative purposes. However, it should be understood that this specification is not limited to the exact arrangement and means of the embodiments shown in the drawings. [Figure 1A] Figures 1A-1B: Recombination of attP and attB sites in cssDNA by Bxb1 integrase according to several embodiments. Figure 1A: Schematic diagram of integrase-mediated recombination of double-stranded circular DNA or single-stranded circular DNA containing the recognized attP and attB sites. Partially double-stranded DNA was prepared by annealing cssDNA with two complementary annealing oligos for the attP and attB sites. Bxb1 integrase is expected to catalyze the intrinsic recombination between the attP and attB sites to produce attL and attR, thereby resulting in two minicircles. Forward and reverse primers were designed to face outwards. Once the recombination of attP and attB occurs, the forward and reverse primers amplify a 250 bp band from the resulting minicircle containing the attL site. [Figure 1B]Figures 1A-1B: Recombination of attP and attB sites in cssDNA by Bxb1 integrase according to several embodiments. Figure 1B: Circular DNA (dsDNA, cssDNA, or partially double-stranded cssDNA) was delivered to K562 cells with or without Bxb1 mRNA. 24 hours after electroporation, the total DNA was isolated and the presence of minicircles was determined by PCR. Top: Minicircles were detected when using double-stranded phagemid DNA substrate. Bottom: While trace amounts of minicircles were detected when cssDNA was used as the integrase substrate, a significant amount of minicircles was detected when using hybrid DNA (cssDNA with double strands at the attP and attB sites) as the Bxb1 integrase. [Figure 2A] Figures 2A-2B: Genomic manipulation of iPSC cells using integrase and partially double-stranded cssDNA according to several embodiments. Figure 2A: Schematic diagram of a study design to investigate integrase-mediated genome editing at the Rab11a locus in human cells. Cas9 nuclease was guided to the Rab11a target site by gRNA to induce a double-strand break. Insertion of a "landing pad" at the target site was achieved using an oligo containing attB sequences flanked by 50 nt homology arms on both sides of the DNA break site as an HDR repair template. An EF1a promoter-driven GFP DNA cargo was co-delivered with Bxb1 mRNA following the attP site, and the efficiency of integrase-mediated GFP DNA cargo integration was investigated. Double-stranded phagemids containing the attP site were used as a control. cssDNA was used either directly or to form partially double-stranded cssDNA by annealing with a complementary attB oligo. [Figure 2B]Figures 2A-2B: Genomic manipulation of iPSC cells using integrase and partially double-stranded cssDNA according to several configurations. Figure 2B: Cultured iPSCs were electroporated with Rab11a-RNP, Rab11a-attB oligo, and Bxb1 mRNA along with cargo DNA substrates of different forms (dsDNA, cssDNA, or partially double-stranded DNA). Fifteen days after electroporation, iPSCs exhibiting stable GFP expression were identified by flow cytometry. [Figure 3] Figure 3 shows that the recombinase-based gene editing strategies described herein also function when transposases are used as recombinases, according to several embodiments. K562 cells were either mock-transfected or transfected with cssDNA alone containing the EF1a-GFP coding sequence adjacent to the transposase-recognized inverted terminal repeat (ITR), or with cssDNA and codon-optimized NLS-tagged PiggyBac encoding mRNA. GFP expression was detected in cells 21 days after transfection. Approximately 39.30% GFP reporter expression was observed from stably integrated EF1a-GFP transgenes 21 days after electroporation, compared to only about 2.37% GFP expression from the cssDNA donor template alone, indicating substantially stable genomic integration of the reporter transgene by the NLS-tagged PiggyBac transposase. [Figure 4]Figure 4 shows that, according to several embodiments, recombinase-based gene editing strategies can edit primary T cells when transposases are used as recombinases. Primary T cells were transfected with cssDNA alone containing an EF1a-GFP coding sequence adjacent to a transposase-recognized inverted terminal repeat (ITR), or with cssDNA and codon-optimized NLS-tagged PiggyBac encoding mRNA. Fourteen days after electroporation, approximately 7.54% GFP reporter expression was observed from stably integrated EF1a-GFP transgenes, compared to only approximately 0.16% GFP expression from the cssDNA donor template alone group, indicating substantially stable genomic integration of the reporter transgene by the NLS-tagged PiggyBac transposase. [Modes for carrying out the invention]

[0058] Detailed explanation The following disclosure provides numerous different modes or examples for implementing different features of the subject matter provided. For the sake of brevity of this disclosure, specific examples of components and arrangements are described below. Naturally, these are merely examples and are not intended to be limiting. For example, in the following description, covering or forming a first feature on a second feature may include modes in which the first and second features are formed in direct contact, and also modes in which an additional feature may be formed between the first and second features so that the first and second features do not have to be in direct contact. Furthermore, this disclosure may repeat reference numerals and / or letters in various examples. This repetition is for the purpose of simplification and clarification and does not in itself determine the relationships between the various modes and / or three-dimensional structures discussed.

[0059] While recombinases used in gene editing are not known to recognize single-stranded DNA (ssDNA) molecules or to incorporate ssDNA molecules into double-stranded host DNA (e.g., genomic DNA or mitochondrial DNA), this study made the unexpected discovery that these recombinases can incorporate circular single-stranded DNA (cssDNA) molecules containing a recombinase recognition sequence into host DNA.

[0060] Most unexpectedly, although the efficiency of this cssDNA integration is relatively low, it can be significantly improved by the presence of a single-stranded oligonucleotide that doubles with the cssDNA molecule. For example, in the presence of a double-stranded single-stranded oligonucleotide, the non-restrictive exemplary recombinase Bxb1 was able to achieve a surprisingly high integration efficiency using cssDNA molecules, substantially exceeding the integration efficiency using the corresponding circular double-stranded DNA molecule.

[0061] While we do not wish to be bound by theory, it is hypothesized that after a cssDNA molecule enters a host cell, it passes through the cytoplasm into the nucleus, where cellular mechanisms within the nucleus (such as DNA replication) convert the cssDNA into a circular double-stranded DNA molecule. Recombinase then integrates the newly generated double-stranded DNA molecule into the genomic DNA. The presence of single-stranded oligonucleotides for double-stranding provides primers to these cellular mechanisms, accelerating the conversion of cssDNA to the corresponding double-stranded DNA and thus improving editing efficiency. Furthermore, since both the cssDNA molecule for integration and the single-stranded oligonucleotides for double-stranding pass through the cytoplasm as single-stranded DNA molecules, they do not trigger a host cell alert or activate innate immunity as invading cytoplasmic DNA, which can at least partially explain the remarkably high editing efficiency.

[0062] Therefore, in some respects, the present invention is directed toward cssDNA molecules, such as cssDNA molecules, that are suitable for integration into host DNA by recombinases. CssDNA molecules are relatively more difficult to edit than double-stranded DNA molecules. For this reason, cssDNA molecules are often edited as double-stranded DNA molecules and then converted into cssDNA molecules.

[0063] Therefore, in some aspects, the present invention is directed toward double-stranded DNA molecules, such as double-stranded DNA molecules configured to produce cssDNA molecules as herein. In some aspects, the present invention is directed toward, for example, a kit for producing cssDNA molecules as herein from double-stranded DNA molecules as herein.

[0064] When the cssDNA molecules described herein are appropriately combined with a recombinase and / or a double-stranded oligonucleotide, they can be used to edit host DNA, such as genomic DNA or mitochondrial DNA, in host cells.

[0065] Therefore, in some respects, the present invention is directed toward a kit for performing genome editing.

[0066] The cssDNA molecules described herein, when appropriately combined with a recombinase and / or a double-stranded oligonucleotide, as well as a suitable host cell, can be used to generate genetically modified cells.

[0067] Therefore, in some respects, the present invention is directed toward a system for preparing genetically modified cells.

[0068] definition Where used herein, each of the following terms has the same meaning as it has in this section. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art in which this disclosure belongs. Generally, the nomenclature and experimental procedures in animal pharmacology, pharmaceuticals, peptide chemistry, and organic chemistry used herein are well known and commonly used in the art. It should be understood that the order of steps or the order in which certain actions are performed is not important as long as this disclosure remains practicable. The use of any section heading is intended to aid in the reading of this document and should not be construed as an limitation; information related to a section heading may exist within or outside that particular section. All publications, patents, and patent documents referenced herein are incorporated herein by reference in whole as they would be incorporated individually by reference.

[0069] Wherever it is stated in this specification that an element or component is included in and / or selected from a list of elements or components described therein, it should be understood that the element or component may be any one of the elements or components described and may be selected from a group of two or more of the elements or components described therein.

[0070] In the methods described herein, unless a temporal or operational order is explicitly stated, the operations may be performed in any order. Furthermore, certain operations may be performed simultaneously unless they are explicitly stated to be performed separately in the claim language. For example, the claimed operation of performing X and the claimed operation of performing Y may be performed simultaneously within a single operation, and the resulting process falls within the literal scope of the claimed process.

[0071] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless otherwise indicated by the context. The term “or” is used to mean an inclusive “or” unless otherwise indicated. The phrases “at least one of A and B” or “at least one of A or B” are synonymous with “A, B, or A and B.”

[0072] As used herein when referring to measurable values ​​such as quantities or time periods, “about” means to include variations of ±20% or ±10%, ±5% in a particular embodiment, ±1% in a particular embodiment, or ±0.1% in a particular embodiment from the specified value, insofar as such variations are appropriate for performing the disclosed method.

[0073] Circular single-stranded DNA molecule In several aspects, the present invention is directed toward circular single-stranded DNA (cssDNA) molecules.

[0074] In some embodiments, the cssDNA molecule is configured to be incorporated into a host DNA molecule, such as a host cell's genomic DNA molecule or mitochondrial DNA molecule, by a recombinase.

[0075] In some embodiments, cssDNA molecules contain a recombinase recognition sequence.

[0076] In some embodiments, the term “recombinase” refers to an enzyme capable of forming a complex between a circular double-stranded DNA molecule and a DNA molecule such as the host cell’s genomic DNA or mitochondrial DNA, and incorporating the circular double-stranded DNA into the host DNA. In some embodiments, the recombinase specifically recognizes a recombinase recognition site, which “grasps” the circular double-stranded DNA molecule and transports the double-stranded DNA molecule into the host DNA.

[0077] In some embodiments, the recombinases described herein do not actually directly recognize single-stranded recombinase-recognition sequences. Rather, the recombinase recognizes the recombinase-recognition sequence only when it has been converted to a double-stranded DNA sequence. In some embodiments, the recombinase-recognition sequence in the cssDNA molecule is one of the strands of the corresponding double-stranded DNA recombinase-recognition sequence that is recognizable by the recombinase.

[0078] In some embodiments, recombinases are nonspecific recombinases. In some embodiments, the term “nonspecific recombinase” refers to a recombinase that specifically recognizes a recombinase recognition site on a circular double-stranded DNA molecule but does not recognize a specific sequence on host DNA. Non-limiting examples of nonspecific recombinases include most existing natural transposases, which may or may not have a preference for host DNA sequences for integration but lack specificity for any particular DNA sequence.

[0079] In some embodiments, recombinases are site-specific recombinases (SSRs). In some embodiments, site-specific recombinases specifically recognize both a recombinase recognition site on a circular double-stranded DNA molecule and a specific sequence on host DNA. Thus, site-specific recombinases can incorporate circular double-stranded DNA molecules at specific locations on host DNA. In some embodiments, site-specific recombinases can be classified into tyrosine integrases, serine integrases, and genetically engineered site-specific transposases.

[0080] Non-limiting examples of site-specific recombinases are Cre, Flp, Dre, SCre, VCre, Vika, B2, B3, KD, ΦC31, Bxb1, RDF, λ, HK022, HP1, γδ, ParA, Gin, R4, TP901-1, TG1, PhiRv1, PhiBT 1, SprA, XisF, TnpX, R, A118, spoIVCA, MR11, SCCmec, TndX, XerC, XerD, XisA, Hin, Cin, mrpA, beta, PhiFC1, Fre, Clp, sTre, FimE, HbiF, φFC1, φC1, q This includes retrotransposases coded by 370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, R2, L1, Tol2 Tc1, Tc3, Mariner(Himar 1), Mariner(mos 1), Minos, R1, R2, R3, R4, R5, etc. Non-limiting examples of site-specific recombinases further include modified or manipulated transposases that retain site specificity, such as modified or manipulated Mu transposase, Tn3 transposase, Tn5 transposase, Tn7 transposase, Tol2, Sleeping Beauty transposase, or PiggyBac transposase.

[0081] In some aspects, the recombinase recognition sequence in the cssDNA molecule is the recognition sequence for site-directed recombinase (SSR).

[0082] In some embodiments, cssDNA molecules can be incorporated into DNA molecules, such as genomic DNA molecules or mitochondrial DNA molecules of host cells, in a site-specific manner or similar, with the help of recombinases such as site-directed recombinases.

[0083] In some embodiments, recombinases, such as site-directed recombinases, recognize recombinase recognition sequences in cssDNA molecules (or dsDNA molecules converted from cssDNA molecules).

[0084] In some embodiments, recombinases, such as site-directed recombinases, recognize both a recombinase-recognition sequence in a cssDNA molecule (or a dsDNA molecule converted from a cssDNA molecule) and a "landing pad" sequence in the DNA molecule in the host cell.

[0085] In some embodiments, a recombinase, such as a site-directed recombinase, "grabs" a cssDNA molecule (or a dsDNA molecule converted from a cssDNA molecule) by a recombinase recognition sequence, carries the cssDNA molecule (or the dsDNA molecule converted from a cssDNA molecule) to the immediate vicinity of the host cell DNA molecule using a landing pad sequence as an anchor, and integrates the cssDNA (or the dsDNA molecule converted from a cssDNA molecule) into the host cell DNA in a site-directed manner.

[0086] In some embodiments, the recombinase recognition sequence is an X-over P1 locus (loxP) sequence, a flippase recognition target (FRT) sequence, a rox sequence, an attachment phage (attP) sequence, an attachment bacterium (attB) sequence, an attachment left (attL) sequence, an attachment right (attR) sequence, a VloxP sequence, a vox sequence, or a transposase recognition inverted terminal repeat (ITR). In some embodiments, the recombinase recognition sequence is a sense strand, an antisense strand, or a combination of both.

[0087] In some embodiments, the cssDNA molecule further includes a payload sequence.

[0088] In some embodiments, the payload sequence encodes an RNA molecule or a polypeptide.

[0089] In some embodiments, the payload sequence in a cssDNA molecule is a sense strand, an antisense strand, or a combination of both.

[0090] In some embodiments, the length of the payload sequence ranges from about 1 nucleotide to about 50,000 nucleotides, for example, about 10 nucleotides to about 20,000 nucleotides, about 50 nucleotides to about 10,000 nucleotides, about 100 nucleotides to about 5,000 nucleotides, or about 200 nucleotides to about 2,000 nucleotides. In some embodiments, the length of the payload sequence is about 1 nucleotide, about 10 nucleotides, about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, about 200 nucleotides, about 500 nucleotides, about 1,000 nucleotides, about 2,000 nucleotides, about 5,000 nucleotides, about 10,000 nucleotides, about 20,000 nucleotides, about 50,000 nucleotides, or any range in between.

[0091] In some embodiments, the cssDNA molecules are purified or concentrated. In some embodiments, the cssDNA molecules are considered purified or concentrated if the molar ratio of cssDNA molecules to total DNA molecules in the system is about 50% or higher, for example, about 60% or higher, about 70% or higher, about 80% or higher, about 90% or higher, about 95% or higher, about 98% or higher, or about 99% or higher.

[0092] In some embodiments, cssDNA is synthesized chemically or enzymatically, for example, in vitro. In some embodiments, cssDNA is prepared from one or more double-stranded DNA molecules, for example, using the M13 phage system.

[0093] Kit containing double-stranded DNA molecules for preparing double-stranded DNA molecules and cssDNA. In several aspects, the present invention is directed towards double-stranded DNA (dsDNA) molecules.

[0094] In some embodiments, the dsDNA molecule is a DNA molecule for preparing cssDNA as described herein.

[0095] In some embodiments, a dsDNA molecule includes a template sequence encoding cssDNA and a cssDNA conversion element for converting the dsDNA molecule into a cssDNA molecule.

[0096] In some embodiments, the double-stranded DNA molecule is a linear dsDNA molecule or a circular dsDNA molecule.

[0097] In some embodiments, the cssDNA conversion sequence includes a packing signal arising from or derived from the origin of replication and / or the genomic DNA of the cssDNA virus, e.g., a DNA packaging element derived from the genomic DNA of a filamentous bacteriophage. While in an infected host cell, cssDNA viral proteins, such as filamentous bacteriophage proteins, replicate the circular single-stranded genomic DNA by recognizing the origin of replication and package the circular single-stranded genomic DNA by recognizing the packing signal. Such mechanisms have been used to mass-produce cssDNA from double-stranded DNA molecules (see, e.g., Xie et al., bioRxiv 2022.12.01.518578, Cha et al., Advanced Functional Materials Volume 31, Issue 35, August 26, 2021, and Shepherd et al., Sci Rep 9, 6121 (2019)). Therefore, in some embodiments, the cssDNA conversion sequence includes a filamentous bacteriophage replication origin or packing signal.

[0098] In some embodiments, the cssDNA conversion sequence includes an M13 phage f1 origin, an M13 phage replication initiator, an M13 phage replication terminator, or an M13 phage packing signal (PS).

[0099] In some embodiments, the dsDNA molecule further comprises a payload sequence, or a payload insertion site for inserting a payload sequence. In some embodiments, the payload sequence is the same as or similar to those described for the cssDNA molecule, but here it is double-stranded.

[0100] In some embodiments, the payload insertion site is a multiple cloning site, such as a multiple cloning site containing restriction enzyme digestion sequences similar to those found in commercially available plasmid vectors.

[0101] As described by Shepherd et al. (Sci Rep9, 6121 (2019)), the construction of a cssDNA molecule may involve the use of multiple double-stranded DNA molecules; therefore, it is worth noting that the dsDNA molecule as used herein does not necessarily have to be a single dsDNA molecule. Accordingly, in some embodiments, the dsDNA molecule is either a single DNA molecule or two or more distinct DNA molecules.

[0102] In some respects, the present invention is directed toward a kit for preparing cssDNA molecules as described herein.

[0103] In some embodiments, the kit includes the dsDNA molecule as defined herein, and components for converting the dsDNA molecule to cssDNA. Non-limiting examples of such components include the M13 helper plasmid (see, for example, Xie et al., bioRxiv 2022.12.01.518578).

[0104] kit for performing genome editing In some aspects, the present invention is directed toward a kit for performing genome editing in cells. In some embodiments, genome editing involves editing the genomic DNA or mitochondrial DNA of a host cell, such as a mammalian cell, a plant cell, or another type of eukaryotic cell, or a bacterial cell.

[0105] In some embodiments, the kit comprises a cssDNA molecule as defined herein, a dsDNA molecule as defined herein, or a kit for preparing a cssDNA molecule as defined herein, and a recombinase or nucleic acid encoding a recombinase.

[0106] In some embodiments, a recombinase recognizes a recombinase-recognition sequence in cssDNA. In some embodiments, a recombinase recognizes a recombinase-recognition sequence in cssDNA (or circular double-stranded DNA converted from cssDNA by the host cell) and inserts the cssDNA (or circular double-stranded DNA converted from cssDNA by the host cell) into host DNA. In some embodiments, a recombinase (such as a site-directed recombinase) recognizes both the recombinase-recognition sequence in cssDNA (or circular double-stranded DNA converted from cssDNA by the host cell) and a "landing pad" sequence on the host DNA, and uses the landing pad sequence as an anchor point to insert the cssDNA (or circular double-stranded DNA converted from cssDNA by the host cell) into the host DNA.

[0107] In some embodiments, the recombinase is the same as or similar to those described elsewhere in this specification. In some embodiments, the recombinase is a serine integrase, tyrosine integrase, or transposase (such as a site-specific transposase). In some embodiments, the recombinase is Cre, Flop, Dre, SCre, VCre, Vika, B2, B3, KD, ΦC31, Bxb1, RDF, λ, HK022, HP1, γδ, ParA, Gin, R4, TP901-1, TG1, PhiRv1, PhiBT1, SprA, XisF, TnpX, R, A118, spoIVCA, MR11, SCCmec, TndX, XerC, XerD, XisA, Hin, Cin, mrpA, beta, PhiFC1, Fre, Clp, sTre, FimE, HbiF, φFC1, φC1, q Retrotransposases coded by 370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, R2, L1, Tol2 Tc1, Tc3, Mariner(Himar 1), Mariner(mos 1), Minos, R1, R2, R3, R4, R5, etc. In some embodiments, recombinases include Mu transposase, Tn3 transposase, Tn5 transposase, Tn7 transposase, Tol2, Sleeping Beauty transposase, and PiggyBac transposase. Naturally occurring transposases are usually not site-specific. However, transposases can become site-specific through modification and / or manipulation.

[0108] In some embodiments, the kit comprises a nucleic acid encoding a recombinase, which is mRNA, single-stranded DNA, or double-stranded DNA. In some embodiments, the nucleic acid encoding the recombinase is incorporated into the cssDNA molecule as described herein. In some embodiments, the nucleic acid encoding the recombinase is not incorporated into the cssDNA molecule as described herein, but is located on a separate polynucleotide molecule.

[0109] In some embodiments, the kit further comprises an oligonucleotide that is fully or partially complementary to the cssDNA molecule. In some embodiments, the oligonucleotide is DNA, RNA, a DNA-RNA hybrid, or a chemically modified nucleic acid.

[0110] In some embodiments, oligonucleotides and cssDNA have complementarity of about 80% or more, for example, about 85% or more, about 90% or more, about 95% or more, about 98% or more, or 100% (i.e., the oligonucleotide is perfectly complementary to the cssDNA). As used herein, an oligonucleotide shorter than cssDNA is 80% complementary to cssDNA if 80% of the nucleotides in the oligonucleotide are complementary to the cssDNA, and the oligonucleotide is perfectly complementary to cssDNA if all of the nucleotides in the oligonucleotide are complementary to the cssDNA.

[0111] In some embodiments, the length of a nucleotide ranges from about 5 nucleotides to about 200 nucleotides, for example, about 8 nucleotides to about 160 nucleotides, about 10 nucleotides to about 120 nucleotides, about 15 nucleotides to about 100 nucleotides, or about 20 nucleotides to about 80 nucleotides. In some embodiments, the length of a nucleotide is about 5 nucleotides, about 8 nucleotides, about 10 nucleotides, about 12 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, about 100 nucleotides, about 120 nucleotides, about 140 nucleotides, about 160 nucleotides, about 180 nucleotides, about 200 nucleotides, or any range in between.

[0112] In some embodiments, oligonucleotides are primers for converting cssDNA molecules to circular double-stranded DNA molecules in cells. In some embodiments, oligonucleotides act as primers for the DNA replication mechanism in a host cell, for example, in the nucleus of a cell, so that the DNA replication mechanism can more easily convert cssDNA molecules to circular double-stranded DNA molecules.

[0113] In some embodiments, the kit comprises a nucleic acid encoding a recombinase, the nucleic acid encoding the recombinase being part of a cssDNA molecule or a dsDNA molecule.

[0114] In some embodiments, the kit further includes components for introducing cssDNA and a recombinase or nucleic acid encoding the recombinase into cells. Non-limiting examples of such components include lipid nanoparticles, metal nanoparticles, micelles, microinjectors, electroporation cuvettes, exosomes, and the like.

[0115] In some embodiments, the recombinase and the recombinase recognition sequence are matched such that the recombinase recognizes both the recombinase recognition sequence and can incorporate the cssDNA molecule (or its double-stranded conversion product produced by the host cell) into the host DNA.

[0116] System for preparing genetically modified cells In several aspects, the present invention is directed towards a system for preparing genetically modified cells.

[0117] In some embodiments, the system comprises a kit for performing genome editing and cells to be manipulated.

[0118] In some embodiments, the cellular genomic DNA or mitochondrial DNA contains landing pad sequences for the incorporation of cssDNA molecules (or double-stranded conversion products produced by the host cell).

[0119] In some embodiments, the landing pad sequence is recognized by a recombinase, which then cleaves the cssDNA molecule and inserts the linearized cssDNA molecule (or a double-strand conversion product produced by the host cell) into genomic DNA or mitochondrial DNA using the landing pad sequence as an anchor site.

[0120] In some embodiments, the landing pad sequence is an X-over P1 locus (loxP) sequence, a flippase recognition target (FRT) sequence, a rox sequence, an attachment phage (attP) sequence, an attachment bacterium (attB) sequence, an attachment left (attL) sequence, an attachment right (attR) sequence, a VloxP sequence, a vox sequence, or a transposase recognition inverted terminal repeat (ITR) sequence.

[0121] In some embodiments, the “landing pad” sequence (i.e., the targeted genomic site for integration) is any site recognized by the recombinase herein.

[0122] In some embodiments, the “landing pad” sequence can be an insertion into host DNA or a locus of a mutated gene.

[0123] In some embodiments, the “landing pad” is inserted into host DNA via a site-specific nuclease selected from a group including Cas nucleases, zinc finger nucleases (ZFNs), meganucleases, and transcription activator-like effector nucleases (TALENs). In some embodiments, the “landing pad” sequence is inserted via a guided prime editor technique without double-strand breaks. Non-limiting examples of prime editor techniques include those using Cas 9 H840A nickase fused with reverse transcriptase (RT) (Cas9n(H840A)), where genome editing is achieved using prime editing guide RNA (pegRNA), etc.

[0124] In some embodiments, the cells are bacterial cells, plant cells, or mammalian cells.

[0125] In some embodiments, the cells are isolated cells, cells derived from a cell line, primary cells, or cells under study.

[0126] In some embodiments, the cells are pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), B cells, T cells, natural killer (NK) cells, or terminally differentiated non-dividing cells such as neurons, muscle cells, or hepatocytes.

[0127] In some embodiments, the recombinase, the recombinase recognition sequence, and the “landing pad” sequence are matched such that the recombinase recognizes both the recombinase recognition sequence and the “landing pad” sequence and can incorporate the cssDNA molecule (or its double-stranded conversion product produced by the host cell) into the host DNA.

[0128] Methods for performing genetic manipulation In some embodiments, the present invention is directed toward a method of performing genetic manipulation in cells, for example, editing the genomic DNA or mitochondrial DNA of a cell by inserting a cssDNA molecule (or a double-stranded conversion product thereof produced by the cell) into the DNA of the cell.

[0129] In some embodiments, the method includes the step of introducing the cssDNA molecule described herein and a recombinase or nucleic acid encoding a recombinase into a cell.

[0130] In some embodiments, the recombinase recognizes the recombinase-recognition sequence in the cssDNA, cleaves the cssDNA, and inserts it into the cell's genomic DNA or mitochondrial DNA.

[0131] In some embodiments, the recombinase is a serine recombinase, a tyrosine recombinase, or a site-specific transposase. In some embodiments, the recombinase is Cre, Flop, Dre, SCre, VCre, Vika, B2, B3, KD, ΦC31, Bxb1, RDF, λ, HK022, HP1, γδ, ParA, Gin, R4, TP901-1, TG1, PhiRv1, PhiBT1, SprA, XisF, TnpX, R, A118, spoIVCA, MR11, SCCmec, TndX, XerC, XerD, XisA, Hin, Cin, mrpA, beta, PhiFC1, Fre, Clp, sTre, FimE, HbiF, φFC1, φC1, q Retrotransposases coded by 370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, R2, L1, Tol2 Tc1, Tc3, Mariner(Himar 1), Mariner(mos 1) Minos, R1, R2, R3, R4, R5, Mu transposase, Tn3 transposase, Tn5 transposase, Tn7 transposase, Tol2, Sleeping Beauty transposase, or PiggyBac transposase.

[0132] In some embodiments, the method further includes the step of introducing into cells an oligonucleotide that is fully or partially complementary to the cssDNA molecule, such as a double-stranded oligonucleotide described elsewhere in this specification.

[0133] In some embodiments, the cellular mechanisms of a cell use oligonucleotides as primers to convert cssDNA molecules into circular double-stranded DNA molecules.

[0134] In some embodiments, the method further includes the step of introducing either the sense strand or the antisense strand cssDNA, or both the sense strand and the antisense strand cssDNA, into cells. In some embodiments, only the sense strand cssDNA is introduced. In some embodiments, only the antisense strand is introduced. In some embodiments, both the sense strand and the antisense strand are introduced.

[0135] In some embodiments, the cellular genomic DNA or mitochondrial DNA includes a landing pad sequence for the incorporation of cssDNA molecules. In some embodiments, the cells, the "landing pad" sequence, and / or the method of introducing the "landing pad" sequence into the cells are the same as or similar to those described elsewhere in this specification.

[0136] In some embodiments, the landing pad sequence is recognized by a recombinase, which then cleaves the cssDNA molecule and inserts the linearized cssDNA molecule into genomic DNA or mitochondrial DNA using the landing pad sequence as an anchor site.

[0137] In some embodiments, the landing pad sequence is an X-over P1 locus (loxP) sequence, a flippase recognition target (FRT) sequence, a rox sequence, an attachment phage (attP) sequence, an attachment bacterium (attB) sequence, an attachment left (attL) sequence, an attachment right (attR) sequence, a VloxP sequence, a vox sequence, or a transposase recognition inverted terminal repeat (ITR) sequence.

[0138] In some embodiments, the cells are bacterial cells, plant cells, or mammalian cells.

[0139] In some embodiments, the cells are isolated cells, cells derived from a cell line, primary cells, or cells under study.

[0140] In some embodiments, the cells are pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), B cells, T cells, natural killer (NK) cells, or terminally differentiated non-dividing cells such as neurons, muscle cells, or hepatocytes. [Examples]

[0141] This specification is further described by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, this specification should not be construed as being limited in any way to the following examples, but rather as encompassing any and all variations that become apparent as a result of the disclosures provided herein.

[0142] Example 1: Integrase-mediated genome manipulation using a partially double-stranded circular single-stranded DNA as a donor template. DNA manipulation is paramount in synthetic biology. Genome editing tools are rapidly expanding and being rapidly adapted in basic research and therapeutic applications. As the human genome and the pathogenesis of various diseases become more comprehensively understood, genetic engineering has emerged as a powerful tool for treating diseases and conducting biomedical research. Among the many methods for genetic engineering, integrase technology has been well studied, but its full potential is not yet being utilized. Integrases are site-directed recombinases (SSRs) involved in the integration of DNA into the host cell genome. They recognize short DNA sequences unique to both the host genome and foreign DNA species, and create hybrid sequences by inserting transgenes into the host genome.

[0143] Serine integrases, a subfamily of integrases, are encoded by lysogenic bacteriophages and catalyze their integration into bacterial genomes through the recombination of attP (phage) and attB (bacteria) attachment sites, thereby creating attL (left) and attR (right) sites. Importantly, the recombination of attP and attB is highly directional and reversible only in the presence of a single accessory protein called a recombination-directing factor (RDF), thereby inducing permanent changes in the genome and making them valuable tools for genetic manipulation. Recombination by serine integrases typically requires only an integrase protein and a small att site (approximately 50 bp), making these proteins powerful tools for genome manipulation (Merrick et al., ACS Synth Biol, 7(2), 299-310, 2018).

[0144] Serine integrases were first reported to manipulate the Streptomyces genome at the endogenous attB site (Boccard et al., Plasmid, 21(1), 59-70, 1989; Kuhstoss et al., Gene, 97(1), 143-146, 1991). These enzymes have been widely used to manipulate many organs, including Chinese hamster ovary (CHO) cells (Andreas et al., Nucleic Acids Res, 30(11), 2299-2306, 2002) and Drosophila embryos (Groth et al., Genetics, 166(4), 1775-1782, 2004). Genomic insertion of transgenes using serine integrases offers many advantages compared to other genome manipulation methods. In comparison with homologous recombination-based methods, including those using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and homing endonucleases such as CRISPER-Cas9, DNA insertion is mediated by a single enzyme independently of host factors (Gaj et al., Trends Biotechnol, 31(7), 397-405, 2013). Unlike transposons and retroviruses, insertion can be targeted to specific loci known to have the least positional effect on transgene expression (Vrljicak et al., G3 (Bethesda), 6(4), 805-817, 2016). Transgenes integrated by serine integrases via attP×attB recombination are unidirectional and cannot be inverted or re-transposed without the presence of a congeneral RDF. Genome editing approaches using serine integrases can be broadly classified into three categories: those that integrate DNA into existing genomic loci (pseudo-sites), those that use "landing pads" (single att sites integrated into the genome), and those that use cassette exchange via recombinases.

[0145] To date, phages PhiC31 (Andreas et al., Nucleic Acids Res, 30(11), 2299-2306, 2002), PhiBT1 (Xu et al., Nucleic Acids Res, 36(1), e9, 2008), Bxb1 (Russell et al., Biotechniques, 40(4), 460, 462, 464, 2006; Yamaguchi et al., PLoS One, 6(2), e17267, 2011), and R4 (Olivares et al., Gene, 278(1-2), 167-176, 2001; Yamaguchi et al., PLoS One, 6(2), e17267) have been identified. Four serine integrases derived from 2011 have been shown to facilitate site-specific integration of DNA into the mammalian genome. Of these, PhiC31 and Bxb1 integrases are widely used in genetic engineering and biotechnology research. Both can insert longer DNA sequences than other editing tools can, but require the presence of an attB site as a “landing pad.” Current strategies using integrases for gene editing involve first inserting the attB site into the genome at the desired location, e.g., AAVS1. This step is typically performed by a nuclease such as Cas9 (or a modified version thereof). Next, a plasmid containing the transgene sequence and attP is introduced into the cell along with PhiC31 or Bxb1 integrase, and then the transgene is integrated into the host genome at the attB site. In fact, CRISPR prime editing techniques are used to pre-position the necessary “landing pad” in the mammalian cell genome. When combined with serine integrase (i.e., Bxbi integrase), this technology enabled targeted incorporation of DNA exceeding 5 kb (Anzalone et al., Nat Biotechnol, 40(5), 731-740, 2022; Yarnall et al., Nat Biotechnol. 2022 Nov 24).This technology expands the possibilities of precise genome editing by enabling large-scale, multiplexed gene insertions without the need for double-strand breaks (DSBs).

[0146] While integrase-mediated genome manipulation is a powerful tool for editing DNA, it is not without its limitations. Low efficiency is one of the limitations inherent in this technique, meaning that not all cells are successfully edited. This can limit the usefulness of this technique, especially when dealing with cells or tissues that are difficult to manipulate. One way to improve efficiency is to supply a larger volume of cargo DNA substrate. However, site-directed recombinases require circular double-stranded DNA (dsDNA), usually plasmids, as donors for the transgene. The use of dsDNA suffers from high cytotoxicity upon intracellular delivery (especially at higher doses), which leads to significant cell death via innate immune responses mediated primarily by cytoplasmic DNA sensing pathways such as Toll-like receptor 9 (TLR9), cyclic GMP-AMP synthase (cGAS), or interferon gene-stimulating factor (STING) (Briard et al., Physiology (Bethesda), 35(2), 112-124, 2020; Schlee & Hartmann, Nat Rev Immunol, 16(9), 566-580, 2016; Zahid et al., Front Immunol, 11, 613039, 2020). Low cell viability directly impacts the efficiency of gene editing and therefore limits the application of SSR in gene therapy.

[0147] A technological platform has been developed for the clean and scalable purification of circular single-stranded DNA (cssDNA) carrying gene payloads up to 20 kb from engineered phagemids. The homologous directing repair (HDR) donor template of cssDNA has been established to enable higher genomic engineering efficiency. More importantly, cssDNA does not trigger a cellular innate immune response when delivered into cells, thus reducing cytotoxicity compared to its double-stranded DNA template counterpart, demonstrating its applicability to genetic diseases and immunotherapy (Xie et al., 2022). Furthermore, due to its single-stranded structure, cssDNA possesses only half the molecular weight of its double-stranded DNA counterpart. These inherent properties make it possible to achieve a larger molar mass of cssDNA using the same amount of material in genetic engineering for higher efficiency.

[0148] cssDNA has demonstrated to be an excellent donor template for HDR-mediated gene knock-in and was considered suitable for broader applications, such as acting as a DNA substrate for SSRs. Since the recognition sites of SSRs (attP and attB) are thought to be double-stranded, cssDNA may not be the optimal substrate for SSRs. Therefore, the attP or attB site in cssDNA may need to hybridize with a complementary oligo to form a double-stranded site for SSR recognition. Hybrid DNA (cssDNA with partial double-stranding at the integrase recognition site) was hypothesized to be a suitable substrate for SSR-mediated manipulation. If a "landing pad" (e.g., the attB site) is present in the genome, it is expected that integrase will "cut" the transgene payload from the cssDNA molecule and "paste" it into the host genome, and the cell's own repair mechanisms will synthesize a second strand to restore the double-helix structure of the genomic DNA, thus completing permanent gene transfer. Due to the limited double-stranded region (only <50 bp) in hybrid DNA, its cytotoxicity is expected to be lower compared to fully double-stranded dsDNA.

[0149] Example 2: cssDNA having a double-stranded region at the attB and attP sites is an efficient DNA substrate for Bxb1 integrase. This study first designed a double-stranded phagemide with payloads adjacent to the attB and attP sites, and used this phagemide to produce cssDNA. In the purified cssDNA, the antibiotic resistance gene and the *E. coli* replication origin region were removed, while the single-stranded payload with two attB and attP sites was maintained. Partially double-stranded cssDNA was prepared by annealing the cssDNA using two complementary annealing oligos for the attP and attB sites. Codon-optimized Bxb1 mRNA for human cell expression was synthesized by in vitro transcription. Bxb1 is expected to catalyze embedded recombination between the attP and attB sites to produce attL and attR, resulting in two minicircles. Forward and reverse primers were designed to face outwards. Using this design, the amplification products for non-recombinant dsDNA and cssDNA are expected to be 2800 bp and 800 bp, respectively (Figure 1A). Upon recombination of attP and attB, forward and reverse primers amplified a 250 bp band from the resulting minicircle containing the attL site (Figure 1A). Circular DNA (dsDNA, cssDNA, or partially double-stranded cssDNA) was co-electroporated into K562 cells with Bxb1 mRNA. Total DNA was isolated 24 hours after electroporation, and the presence of minicircles was determined by PCR. As expected, when double-stranded phagemid DNA containing attB and attP sites was co-electroporated with Bxb1 mRNA, PCR amplification yielded 2800 bp and 250 bp bands. However, without Bxb1 integrase, only the 2800 bp band was detectable (Figure 1B). When cssDNA was electroporated into K562 cells, only an 800 bp product was amplified by PCR. When cssDNA was co-electroporated with Bxb1 integrase, in addition to the 800 bp band, a faintly visible 250 bp PCR product was also detected.When hybrid DNA (cssDNA with double helix at the attP and attB sites) was co-electroporated with Bxb1 integrase, the 250 bp PCR product became thicker, indicating that attB × attP recombination generated a much larger number of minicircle products. As a negative control, hybrid DNA in the absence of Bxb1 integrase did not yield a 250 bp PCR band (Figure 1B). These results demonstrate that both cssDNA alone, or cssDNA with partially double helix attP and attB sites, are efficient DNA substrates for recombination via Bxb1 integrase.

[0150] Example 3: Efficient genome integration in iPSC cells using integrase and a partially double-stranded cssDNA substrate This study then sought to determine whether cssDNA or its partially double-stranded form could be used as an integrase DNA substrate for genomic manipulation in human cells. The study aimed to knock in the “landing pad” (attB site) at the Rab11a locus in human induced pluripotent stem cells (iPSCs). A highly efficient cleavage-efficient Rab11a gRNA was demonstrated, which can also be used for highly efficient targeted knock-in in multiple human cells. A ribonucleoprotein (RNP) targeting the Rab11a locus, along with a Rab11a-attB oligo (a 46-base attB flanked by 50-base homology arms on both sides), was used for targeted attB landing pad knock-in (Figure 2A). A phagemide with an EF1α promoter-driven GFP payload downstream of the attP site was cloned and used for cssDNA production (Figure 2A). The cssDNA was used either directly or annealed with a complementary attB oligo to form partially double-stranded cssDNA. Cultured iPSCs were co-electroporated with Rab11a-RNP, Rab11a-attB oligo, and Bxb1 mRNA, along with different forms of cargo DNA substrates (dsDNA, cssDNA, or hybrid DNA). Fifteen days after electroporation, iPSCs exhibiting stable GFP expression were identified by flow cytometry. Cells under all conditions were treated with Rab11a RNP and Rab11a-attB oligo. As shown in Figure 2B, no GFP-positive cells were observed in the absence of Bxb1 integrase. In the presence of Bxb1 integrase, the dsDNA substrate resulted in approximately 9% GFP-positive cells, while the cssDNA substrate resulted in less than 1% GFP-positive cells. However, when using the partially double-stranded cssDNA substrate, GFP cargo was integrated into the genome of approximately 30% of the cells. These data demonstrate that cssDNA, which is partially double-stranded and contains a double-stranded region only at the integrase recognition site (attP), is an excellent DNA substrate for efficient cargo DNA integration via integrase.

[0151] To the best of our knowledge, this is the first demonstration of cssDNA or a partially double-stranded form thereof as an integrase substrate for SSR-based genome manipulation. The advantages of cssDNA (e.g., lower cytotoxicity, higher molecular weight efficiency, and gene manipulation efficiency) are fully applicable to further applications in genome manipulation in a homology-independent manner.

[0152] Example 4: Efficient genome integration in K562 cells using transposase and cssDNA donor This study aimed to determine whether cssDNA could be used as a DNA donor substrate for human cell genome manipulation using a transposase system. First, a double-stranded phagemide encoding an EF1a-GFP reporter payload adjacent to an ITR site was designed, and this phagemide was used to produce a cssDNA donor template. In the purified cssDNA, the antibiotic resistance gene and the E. coli origin of replication region were removed, while the single-stranded payload with two ITR sites was maintained. Codon-optimized NLS-tagged PiggyBac transposase mRNA for human cell expression was synthesized by in vitro transcription. The PiggyBac transposase is expected to recognize the conventional dsDNA morphology of the 5' and 3' ITR sites and catalyze the incorporation of the DNA sequence adjacent to the ITR site. Once stable incorporation occurs, GFP reporter expression can be detected for several weeks. In this study, circular DNA containing EF1a-GFP adjacent to the 5' and 3' ITRs was co-electroporated into K562 cells along with NLS-tagged PiggyBac transposase mRNA. 21 days post-electroporation, approximately 39.30% GFP reporter expression was observed from the stably integrated EF1a-GFP transgene, compared to only about 2.37% from the cssDNA donor template alone. This demonstrated substantially stable genomic integration of the reporter transgene by NLS-tagged PiggyBac transposase (Figure 3).

[0153] Example 5: Efficient genome integration in human primary T cells using transposase and cssDNA donor Next, this study aimed to determine whether genomic integration via transposition using a cssDNA donor template is applicable to primary cell types. This study used the same cssDNA EF1a-GFP reporter payload adjacent to the 5' and 3' ITR sites. In the study, circular DNA containing EF1a-GFP adjacent to the 5' and 3' ITRs was co-electroporated with NLS-tagged PiggyBac transposase mRNA into human peripheral blood-derived CD4 / CD8 double-positive T lymphocytes. Fourteen days after electroporation, approximately 7.54% GFP reporter expression was observed from the stably integrated EF1a-GFP transgene, compared to only about 0.16% GFP expression from the cssDNA donor template alone, demonstrating substantially stable genomic integration of the reporter transgene by NLS-tagged PiggyBac transposase (Figure 4). To the best of our knowledge, this is the first cssDNA used as a transposase donor DNA substrate for genomic manipulation. The advantages of cssDNA (e.g., lower cytotoxicity, higher molecular weight efficiency, and genetic manipulation efficiency) make it fully applicable to further applications in genome manipulation in a homology-independent manner.

[0154] Example 6: Materials and Methods Preparation of template circular single-stranded DNA from M13 phage Donor template sequences for the integrase system or transposase were constructed as dsDNA and cloned into phagemide vectors. XL1-Blue E. coli strains were co-transformed with phagemides containing the M13 helper plasmid and double-stranded donor templates and selected on agar plates with kanamycin (50 μg / mL) and carbenicillin (100 μg / mL). Single colonies were selected and osmotically filtered in 250 mL of 2xYT medium (1.6% tryptone, 1% yeast extract, 0.25% NaCl). 600The cells were grown for approximately 24 hours (37°C, 225 rpm) until the concentration reached 2.5-3.0. The bacteria were pelletized by centrifugation, and the phage particles in the supernatant were precipitated using PEG-8000. The precipitated phage particles were then pelletized by centrifugation, washed, and dissolved in 20 mM MOPS, 1 M guanidine-HCl, and 2% Triton X-100. The cssDNA released from the phages was then extracted using the NucleoBond Xtra Midi EF kit (Macherey-Nagel) according to the manufacturer's instructions. The cssDNA concentration was determined using Nanodrop for ssDNA, and the yield was 10 μg per 1 ml of liquid culture. The ratio of absorbance (A) was calculated. 260 nm / A 280 The nm and 260 nm / 230 nm values ​​reflect consistent purity from serial preparations (1.8 and >2, respectively). Recombinant cssDNA is validated by Sanger DNA sequencing using custom-designed staggered sequencing primers for full applicability.

[0155] cell culture K562 (ATCC) cells were maintained in RPMI-1640 medium containing 10% FBS and 1% penicillin and streptomycin. iPSCs (ThermoFisher Scientific) were cultured in complete StemFlex (ThermoFisher Scientific) medium in flasks coated with vitronectin. iPSC colonies were periodically checked and subcultured every 3-4 days using ReLeSR (StemCell Technologies). iPSCs were ready for electroporation after 2-3 subculturing. All cells were maintained in a humidified incubator at 37°C and 5% CO2 unless otherwise specified. Cell viability was determined on a specific day after the procedure using a Via2-Cassette in a NucleoCounter® NC-202 (ChemoMetec).

[0156] Preparation of partially double-stranded cssDNA To prepare cssDNA that is partially double-stranded at the attP and attB sites, reverse-complementary attP and attB oligos (50 pmol each) were mixed with cssDNA (25 pmol) in 1× NEB buffer r2.1. The mixture was incubated at 75°C for 3 minutes and then cooled to room temperature at a ramp rate of 0.1°C / sec. The oligo-annealed cssDNA can be stored at 4°C for future use.

[0157] Electroporation Electroporation of K562, iPSCs, and primary T cells was performed using an Amaxa® 96-well Shuttle® with a 4D Nucleofector (Lonza). 25 picomoles of sNLS-SpCas9-sNLS nuclease (Aldevron) per reaction was used together with 50 pmol of sgRNA (synthesized by Integrated DNA Technologies). The Cas9 nuclease and sgRNA were pre-complexed in a supplemented Nucleofector® solution at room temperature for 20 minutes, and the RNP solution was increased to a final volume (10×) of 2.5 μL per electroporation reaction. For mRNA delivery nucleases, 1 μg of Bxb1 mRNA was co-electroporated with 50 pmol of sgRNA and the indicated amount of dsDNA or cssDNA. For PiggyBac transposase electroporation, 2 ug of mRNA was used together with 2 ug of cssDNA. For electroporation of K562 cells, the SF Cell Line 4D-Nucleofector® kit and 250,000–500,000 cells per reaction were used. For electroporation of iPSC cells, 100,000 cells per reaction were used with the P3 Primary Cell 4D-Nucleofector® kit. After electroporation, the cells were placed in a humidified 32°C incubator with 5% CO2 for 12–24 hours, then transferred to a 37°C incubator for a further 3–10 days. For electroporation of primary human T lymphocytes, primary T cells were isolated and enriched from Leukopak using a MultiMCAS Cell24 Separator Plus (Miltenyi) with a Pan T Cell MicroBead Cocktail, and CD4+ and CD8+ pan T cells were cryopreserved for later use. T cells were cultured and proliferated in TexMACS medium (Miltenyi) supplemented with 200 IU / mL human IL-2 IS (Miltenyi).Prior to electroporation, T cells were activated for 2 days using T Cell TransAct (Miltenyi). Forty-eight hours after the start of T cell initiation and activation, the T cells were electroporated using the Amaxa® 96-well Shuttle® 4D Nucleofector. 2 × 10⁻⁶ cells. 6 Each cell was mixed with 2 µg of PiggyBac transposase in vitro transcription mRNA and 2 µg of ITR-EF1a-GFP-ITR cssDNA. After electroporation, the cells were incubated at 32°C and 5% CO2 for 24 hours. The cells were then washed and transferred to G-Rex 24 multiwell cell culture plates (Wilson Wolf) under standard culture conditions at 37°C and 5% CO2 in IL-2 supplemented TexMACS medium, and replenished every 3–4 days prior to GFP reporter analysis on the day instructed after electroporation.

[0158] Bxb1 mRNA preparation by in vitro transcription Codon-optimized Bxb1 for human cell expression, fused with the SV40 nuclear localization signal at the N-terminus, was cloned into a pGEM-4A vector. A 1 μg linearized DNA template was transcribed in vitro in 20 μl of reaction mixture using 1.5 μl of T7 RNA polymerase mix and 5 mM each of GTP, ATP, CTP, and UTP from the HiScribe T7 mRNA synthesis kit (NEB E2080S), according to the manufacturer's instructions. RNA was purified by Zymo min-spin column and then eluted in 80 μL of nuclease-free water.

[0159] Preparation of PiggyBac transposase mRNA by in vitro transcription Codon-optimized PiggyBac transposase for human cell expression was cloned into a pGEM-4A vector. A 1 μg linearized DNA template was transcribed in vitro in 20 μl of reaction mixture using 1.5 μl of T7 RNA polymerase mix and 5 mM each of GTP, ATP, CTP, and UTP from the HiScribe T7 mRNA synthesis kit (NEB E2080S), according to the manufacturer's instructions. RNA was purified by Zymo min-spin column and then eluted in 80 μL of nuclease-free water.

[0160] DNA extraction and PCR amplification of K562 cells K562 was electroporated with or without Bxb1 mRNA in circular DNA constructs (dsDNA, cssDNA, or partially double-stranded cssDNA). 24 hours after electroporation, total DNA was extracted using the Monarch Genomic DNA Purification Kit (New England BioLabs) according to the manufacturer's instructions. The presence of attB×attP recombination minicircles was detected by PCR amplification using forward and reverse primers.

[0161] Flow cytometry analysis All flow cytometry was performed using an Attune NxT flow cytometer (ThermoFisher Scientific) with a 96-well autosampler. Unless otherwise specified, cells were harvested 4–7 days after electroporation, resuspended in fluorescence-activated cell sorting (FACS) buffer (2% FBS in PBS), and stained with 7-AAD (BioLegend) and the indicated cell surface markers. To obtain comparable viable cell counts across conditions, events were recorded from equivalent fixed volumes for all samples. Data analysis, including exclusion of intracellular debris, singlet gating, and viability staining, was performed using FlowJo_v10.8.0_CL software. Analyzed graphs were created using Prism 9 (GraphPad).

[0162] DNA / RNA sequence information used in this study The DNA and RNA sequences used in this study are shown in Table 1 below.

[0163] [Table 1] TIFF2026515686000002.tif235170TIFF2026515686000003.tif235170TIFF2026515686000004.tif37170

[0164] Listed aspects In some aspects, the present invention is directed to the following non-limiting embodiments: Embodiment 1: A circular single-stranded DNA (cssDNA) molecule containing a recombinase-recognition sequence that is recognized by a recombinase. Embodiment 2: The cssDNA molecule of Embodiment 1, wherein the recombinase is a site-directed recombinase (SSR). Embodiment 3: The cssDNA molecule according to Embodiment 2, wherein the site-specific recombinase is a tyrosine integrase, a serine integrase, or a site-specific transposase. Embodiment 4: The cssDNA molecule of Embodiment 3, wherein the recombinase recognition sequence is an X-over P1 locus (loxP) sequence, a flippase recognition target (FRT) sequence, a rox sequence, an attachment phage (attP) sequence, an attachment bacterium (attB) sequence, an attachment left (attL) sequence, an attachment right (attR) sequence, a VloxP sequence, a vox sequence, or a transposase recognition inverted terminal repeat (ITR). Embodiment 5: A cssDNA molecule comprising any one of Embodiments 1 to 4, further comprising a payload sequence. Embodiment 6: The cssDNA molecule of Embodiment 5, wherein the payload sequence encodes an RNA molecule or polypeptide. Embodiment 7: A cssDNA molecule according to any one of Embodiments 1 to 6, wherein the payload sequence contains a length of approximately 1 nucleotide to approximately 50,000 nucleotides. Embodiment 8: A cssDNA molecule from any one of Embodiments 1 to 7, which is either purified or concentrated. Embodiment 9: A template sequence encoding a cssDNA containing a recombinase recognition sequence recognized by a recombinase, cssDNA conversion sequence and A double-stranded DNA (dsDNA) molecule containing [the specified element]. Embodiment 10: The dsDNA molecule of Embodiment 9, which is a linear dsDNA molecule or a circular dsDNA molecule. Embodiment 11: A dsDNA molecule in any one of Embodiments 9 to 10, wherein the cssDNA conversion sequence is a sequence that originates from or is derived from the M13 phage f1 origin, M13 phage replication initiator, M13 phage replication terminator, or M13 phage packing signal (PS). Embodiment 12: A dsDNA molecule comprising any one of Embodiments 9 to 11, further comprising a payload sequence or a payload sequence insertion site for inserting a payload sequence. Embodiment 13: The dsDNA molecule of Embodiment 12, wherein the payload insertion site is a multiplexing site. Embodiment 14: A dsDNA molecule which is one single DNA molecule or two or more distinct DNA molecules, as described in any of Embodiments 9 to 13. Embodiment 15: A cssDNA molecule from any one of Embodiments 1 to 8, or a dsDNA molecule from any one of Embodiments 9 to 14, and a system for converting the dsDNA molecule into the cssDNA, Recombinase or nucleic acid encoding recombinase Includes, The recombinase recognizes the recombinase recognition sequence of the cssDNA. kit. Embodiment 16: The kit according to Embodiment 15, wherein the recombinase is a serine integrase, a tyrosine integrase, or a site-specific transposase. Aspect 17: The recombinase is Cre, Flp, Dre, SCre, VCre, Vika, B2, B3, KD, ΦC31, Bxb1, RDF, λ, HK022, HP1, γδ, ParA, Gin, R4, TP901-1, TG1, PhiRv1, PhiBT1, Spr A, XisF, TnpX, R, A118, spoIVCA, MR11, SCCmec, TndX, XerC, XerD, XisA, Hin, Cin, mrpA, beta, PhiFC1, Fre, Clp, sTre, FimE, HbiF, φFC1, φC1, q Retrotransposases coded by 370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, R2, L1, Tol2 Tc1, Tc3, Mariner(Himar 1), Mariner(mos 1) A kit according to embodiment 15, which is Minos, R1, R2, R3, R4, R5, Mu transposase, Tn3 transposase, Tn5 transposase, Tn7 transposase, Tol2, Sleeping Beauty transposase, or PiggyBac transposase. Embodiment 18: A kit comprising the nucleic acid encoding the recombinase, wherein the nucleic acid encoding the recombinase is mRNA, single-stranded DNA, or double-stranded DNA, as per any one of Embodiments 15 to 17. Embodiment 19: Any one of embodiments 15 to 18 further comprising an oligonucleotide that is completely or partially complementary to the cssDNA molecule. Embodiment 20: The kit according to Embodiment 19, wherein the oligonucleotide is a primer that converts the cssDNA molecule into a circular double-stranded DNA molecule within a cell. Embodiment 21: A kit according to any one of Embodiments 19 to 20, wherein the oligonucleotide comprises a length of approximately 5 nucleotides to approximately 200 nucleotides. Embodiment 22: A kit comprising the nucleic acid encoding the recombinase, wherein the nucleic acid encoding the recombinase is part of the cssDNA molecule or the dsDNA molecule, any one of embodiments 15 to 21. Embodiment 23: Any one of embodiments 15 to 22, further comprising components for introducing the cssDNA and the recombinase or nucleic acid encoding the recombinase into the cell. Embodiment 24: One of the kits from Embodiments 15 to 23, The manipulated cells and A system that includes this. Embodiment 25: The system of Embodiment 24, wherein the genomic DNA or mitochondrial DNA of the cell includes a landing pad sequence for the incorporation of the cssDNA molecule into the genomic DNA or mitochondrial DNA. Embodiment 26: The system of Embodiment 25, wherein the landing pad sequence is recognized by the recombinase, and as a result, the recombinase cleaves the cssDNA molecule and inserts the linearized cssDNA molecule into the genomic DNA or mitochondrial DNA using the landing pad sequence as an anchor site. Embodiment 27: Any one of the systems in Embodiments 25 to 26, wherein the landing pad sequence is an X-over P1 locus (loxP) sequence, a flippase recognition target (FRT) sequence, a rox sequence, an attachment phage (attP) sequence, an attachment bacterium (attB) sequence, an attachment left (attL) sequence, an attachment right (attR) sequence, a VloxP sequence, a vox sequence, or a transposase recognition inverted terminal repeat (ITR). Embodiment 28: A system according to any one of Embodiments 24 to 27, wherein the cells are bacterial cells, plant cells, or mammalian cells. Embodiment 29: Any one of the systems in Embodiments 24 to 28, wherein the cells are isolated cells, cells derived from a cell line, primary cells, or cells under study. Embodiment 30: Any one of the systems in Embodiments 24 to 29, wherein the cells are pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), B cells, T cells, natural killer (NK) cells, or terminally differentiated non-dividing cells such as neurons, muscle cells, or hepatocytes. Embodiment 31: A method for performing genetic manipulation in cells, cssDNA from any one of embodiments 1 to 8, Recombinase or nucleic acid encoding recombinase This includes the step of introducing the following into the cells: The recombinase recognizes the recombinase-recognizing sequence of the cssDNA for incorporating the cssDNA into the cell's genomic DNA or mitochondrial DNA. The aforementioned method. Embodiment 32: The method of Embodiment 31, wherein the recombinase is a serine recombinase, a tyrosine recombinase, or a site-specific transposase. Aspect 33: The recombinase is Cre, Flp, Dre, SCre, VCre, Vika, B2, B3, KD, ΦC31, Bxb1, RDF, λ, HK022, HP1, γδ, ParA, Gin, R4, TP901-1, TG1, PhiRv1, PhiBT1, Spr A, XisF, TnpX, R, A118, spoIVCA, MR11, SCCmec, TndX, XerC, XerD, XisA, Hin, Cin, mrpA, beta, PhiFC1, Fre, Clp, sTre, FimE, HbiF, φFC1, φC1, q Retrotransposases coded by 370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, R2, L1, Tol2 Tc1, Tc3, Mariner(Himar 1), Mariner(mos 1) The method of embodiment 31, wherein the transposase is Minos, R1, R2, R3, R4, R5, Mu transposase, Tn3 transposase, Tn5 transposase, Tn7 transposase, Tol2, Sleeping Beauty transposase, or PiggyBac transposase. Embodiment 34: Any one of embodiments 31 to 33, further comprising the step of introducing into the cell an oligonucleotide that is completely or partially complementary to at least a portion of the cssDNA molecule. Embodiment 35: The method of Embodiment 34, wherein the cellular mechanism of the cell converts the cssDNA molecule into a circular double-stranded DNA molecule using the oligonucleotide as a primer. Embodiment 36: Any one of Embodiments 34 to 35, wherein the oligonucleotide comprises a length of about 5 nucleotides to about 200 nucleotides. Embodiment 37: Any one of embodiments 31 to 36, comprising the step of introducing sense strand cssDNA, antisense strand cssDNA, or a mixture of both into a cell. Embodiment 38: The method of Embodiments 31-37, wherein the genomic DNA or mitochondrial DNA of the cell includes a landing pad sequence for the incorporation of the cssDNA molecule into the genomic DNA or mitochondrial DNA. Embodiment 39: The method of Embodiment 38, further comprising the step of culturing the cells under conditions sufficient to allow the landing pad sequence to be recognized by the recombinase, and sufficient for the recombinase to cleave the cssDNA molecule and insert the linearized cssDNA molecule into the genomic DNA or the mitochondrial DNA using the landing pad sequence as an anchor site. Embodiment 40: Any one of Embodiments 31 to 39, wherein the landing pad sequence is an X-over P1 locus (loxP) sequence, a flippase recognition target (FRT) sequence, a rox sequence, an attachment phage (attP) sequence, an attachment bacterium (attB) sequence, an attachment left (attL) sequence, an attachment right (attR) sequence, a VloxP sequence, a vox sequence, or a transposase recognition inverted terminal repeat (ITR). Embodiment 41: Any one of Embodiments 31 to 40, wherein the cell is a bacterial cell, a plant cell, or a mammalian cell. Embodiment 42: Any one of Embodiments 31 to 41, wherein the cells are isolated cells, cells derived from a cell line, primary cells, or cells under study. Embodiment 43: Any one of Embodiments 31 to 42, wherein the cells are pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), B cells, T cells, natural killer (NK) cells, or terminally differentiated non-dividing cells such as neurons, muscle cells, or hepatocytes.

[0165] To enable those skilled in the art to better understand aspects of this disclosure, several features of the embodiments have been outlined above. Those skilled in the art should acknowledge that this disclosure may readily be used as a basis for designing or modifying other processes and structures to perform the same purposes and / or achieve the same advantages as the embodiments introduced herein. Those skilled in the art should also understand that such equivalent configurations do not deviate from the spirit and scope of this disclosure, and that various changes, substitutions, and modifications may be made herein without deviating from the spirit and scope of this disclosure.

Claims

1. A circular single-stranded DNA (cssDNA) molecule containing a recombinase-recognition sequence that is recognized by recombinase.

2. The cssDNA molecule according to claim 1, wherein the recombinase is a site-directed recombinase (SSR).

3. The cssDNA molecule according to claim 2, wherein the site-specific recombinase is a tyrosine integrase, a serine integrase, or a site-specific transposase.

4. The cssDNA molecule according to claim 3, wherein the recombinase recognition sequence is an X-over P1 locus (loxP) sequence, a flippase recognition target (FRT) sequence, a rox sequence, an attachment-phage (attP) sequence, an attachment-bacteria (attB) sequence, an attachment-left (attL) sequence, an attachment-right (attR) sequence, a VloxP sequence, a vox sequence, or a transposase recognition inverted terminal repeat (ITR).

5. A cssDNA molecule according to any one of claims 1 to 4, further comprising a payload sequence.

6. The cssDNA molecule according to claim 5, wherein the payload sequence encodes an RNA molecule or polypeptide.

7. The cssDNA molecule according to any one of claims 1 to 6, wherein the payload sequence comprises a length of approximately 1 nucleotide to approximately 50,000 nucleotides.

8. A cssDNA molecule according to any one of claims 1 to 7, which is purified or concentrated.

9. A template sequence encoding cssDNA containing a recombinase-recognition sequence recognized by recombinase, cssDNA conversion sequence and A double-stranded DNA (dsDNA) molecule containing [the specified element].

10. The dsDNA molecule according to claim 9, which is a linear dsDNA molecule or a circular dsDNA molecule.

11. The dsDNA molecule according to any one of claims 9 to 10, wherein the cssDNA conversion sequence is a sequence resulting from or derived from an M13 phage f1 origin, an M13 phage replication initiator, an M13 phage replication terminator, or an M13 phage packing signal (PS).

12. A dsDNA molecule according to any one of claims 9 to 11, further comprising a payload sequence or a payload sequence insertion site for inserting a payload sequence.

13. The dsDNA molecule according to claim 12, wherein the payload insertion site is a multiplexing site.

14. A dsDNA molecule according to any one of claims 9 to 13, which is a single DNA molecule or two or more distinct DNA molecules.

15. A cssDNA molecule according to any one of claims 1 to 8, or a dsDNA molecule according to any one of claims 9 to 14, and a system for converting the dsDNA molecule to the cssDNA, Recombinase or nucleic acid encoding recombinase Includes, The recombinase recognizes the recombinase recognition sequence of the cssDNA. kit.

16. The kit according to claim 15, wherein the recombinase is serine integrase, tyrosine integrase, or site-specific transposase.

17. The recombinase is Cre, Flp, Dre, SCre, VCre, Vika, B2, B3, KD, ΦC31, Bxb1, RDF, λ, HK022, HP1, γδ, ParA, Gin, R4, TP901-1, TG1, PhiRv1, PhiBT1, Spr A, XisF, TnpX, R, A118, spoIVCA, MR11, SCCmec, TndX, XerC, XerD, XisA, Hin, Cin, mrpA, beta, PhiFC1, Fre, Clp, sTre, FimE, HbiF, φFC1, φC1, q Retrotransposases coded by 370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, R2, L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1) The kit according to claim 15, wherein the transposase is Minos, R1, R2, R3, R4, R5, Mu transposase, Tn3 transposase, Tn5 transposase, Tn7 transposase, Tol2, Sleeping Beauty transposase, or PiggyBac transposase.

18. The kit according to any one of claims 15 to 17, comprising the nucleic acid encoding the recombinase, wherein the nucleic acid encoding the recombinase is mRNA, single-stranded DNA, or double-stranded DNA.

19. The kit according to any one of claims 15 to 18, further comprising an oligonucleotide that is completely or partially complementary to the cssDNA molecule.

20. The kit according to claim 19, wherein the oligonucleotide is a primer that converts the cssDNA molecule into a circular double-stranded DNA molecule within a cell.

21. The kit according to any one of claims 19 to 20, wherein the oligonucleotide comprises a length of about 5 nucleotides to about 200 nucleotides.

22. The kit according to any one of claims 15 to 21, comprising the nucleic acid encoding the recombinase, wherein the nucleic acid encoding the recombinase is part of the cssDNA molecule or the dsDNA molecule.

23. The kit according to any one of claims 15 to 22, further comprising components for introducing the cssDNA and the recombinase or nucleic acid encoding the recombinase into the cell.

24. A kit according to any one of claims 15 to 23, The manipulated cells and A system that includes this.

25. The system according to claim 24, wherein the genomic DNA or mitochondrial DNA of the cell includes a landing pad sequence for the incorporation of the cssDNA molecule into the genomic DNA or mitochondrial DNA.

26. The system according to claim 25, wherein the landing pad sequence is recognized by the recombinase, and as a result, the recombinase cleaves the cssDNA molecule and inserts the linearized cssDNA molecule into the genomic DNA or mitochondrial DNA using the landing pad sequence as an anchor site.

27. The system according to any one of claims 25 to 26, wherein the landing pad sequence is an X-over P1 locus (loxP) sequence, a flippase recognition target (FRT) sequence, a rox sequence, an attachment phage (attP) sequence, an attachment bacterium (attB) sequence, an attachment left (attL) sequence, an attachment right (attR) sequence, a VloxP sequence, a vox sequence, or a transposase recognition inverted terminal repeat (ITR).

28. The system according to any one of claims 24 to 27, wherein the cells are bacterial cells, plant cells, or mammalian cells.

29. The system according to any one of claims 24 to 28, wherein the cells are isolated cells, cells derived from a cell line, primary cells, or cells under study.

30. The system according to any one of claims 24 to 29, wherein the cells are pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), B cells, T cells, natural killer (NK) cells, or terminally differentiated non-dividing cells such as neurons, muscle cells, or hepatocytes.

31. A method for performing genetic manipulation in cells, cssDNA according to any one of claims 1 to 8, Recombinase or nucleic acid encoding recombinase This includes the step of introducing the following into the cells: The recombinase recognizes the recombinase-recognizing sequence of the cssDNA for incorporating the cssDNA into the cell's genomic DNA or mitochondrial DNA. The aforementioned method.

32. The method according to claim 31, wherein the recombinase is serine recombinase, tyrosine recombinase, or site-specific transposase.

33. The recombinase is Cre, Flp, Dre, SCre, VCre, Vika, B2, B3, KD, ΦC31, Bxb1, RDF, λ, HK022, HP1, γδ, ParA, Gin, R4, TP901-1, TG1, PhiRv1, PhiBT1, Spr A, XisF, TnpX, R, A118, spoIVCA, MR11, SCCmec, TndX, XerC, XerD, XisA, Hin, Cin, mrpA, beta, PhiFC1, Fre, Clp, sTre, FimE, HbiF, φFC1, φC1, q Retrotransposases coded by 370.1, Wβ, BL3, SPBc, K38, Peaches, Veracruz, Rebeuca, Theia, Benedict, KSSJEB, PattyP, Doom, Scowl, Lockley, Switzer, Bob3, Troube, Abrogate, Anglerfish, Sarfire, SkiPole, ConceptII, Museum, Severus, Airmid, Benedict, Hinder, ICleared, Sheen, Mundrea, BxZ2, φRV, R2, L1, Tol2 Tc1, Tc3, Mariner (Himar 1), Mariner (mos 1) The method according to claim 31, wherein the transposase is Minos, R1, R2, R3, R4, R5, Mu transposase, Tn3 transposase, Tn5 transposase, Tn7 transposase, Tol2, Sleeping Beauty transposase, or PiggyBac transposase.

34. The method according to any one of claims 31 to 33, further comprising the step of introducing into the cell an oligonucleotide that is completely or partially complementary to at least a portion of the cssDNA molecule.

35. The method according to claim 34, wherein the cellular mechanism of the cell converts the cssDNA molecule into a circular double-stranded DNA molecule using the oligonucleotide as a primer.

36. The method according to any one of claims 34 to 35, wherein the oligonucleotide comprises a length of about 5 nucleotides to about 200 nucleotides.

37. The method according to any one of claims 31 to 36, comprising the step of introducing sense strand cssDNA, antisense strand cssDNA, or a mixture of both into a cell.

38. The method according to claims 31 to 37, wherein the genomic DNA or mitochondrial DNA of the cell includes a landing pad sequence for the incorporation of the cssDNA molecule into the genomic DNA or mitochondrial DNA.

39. The method according to claim 38, further comprising the step of culturing the cells under conditions sufficient to allow the landing pad sequence to be recognized by the recombinase, and sufficient for the recombinase to cleave the cssDNA molecule and insert the linearized cssDNA molecule into the genomic DNA or mitochondrial DNA using the landing pad sequence as an anchor site.

40. The method according to any one of claims 31 to 39, wherein the landing pad sequence is an X-over P1 locus (loxP) sequence, a flippase recognition target (FRT) sequence, a rox sequence, an attachment phage (attP) sequence, an attachment bacterium (attB) sequence, an attachment left (attL) sequence, an attachment right (attR) sequence, a VloxP sequence, a vox sequence, or a transposase recognition inverted terminal repeat (ITR).

41. The method according to any one of claims 31 to 40, wherein the cell is a bacterial cell, a plant cell, or a mammalian cell.

42. The method according to any one of claims 31 to 41, wherein the cells are isolated cells, cells derived from a cell line, primary cells, or cells in question.

43. The method according to any one of claims 31 to 42, wherein the cells are pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), B cells, T cells, natural killer (NK) cells, or terminally differentiated non-dividing cells such as neurons, muscle cells, or hepatocytes.

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