RNA products and methods of production

Abstract

The invention relates to methods for producing an RNA product (e.g. a guide RNA or prime editing guide RNA (pegRNA)) comprising ligating at least three RNA molecules. The at least three RNA molecules comprise: a first RNA molecule that is a chemically synthesized RNA molecule, a second RNA molecule that is an in vitro transcription (IVT) produced RNA molecule, and a third RNA molecule that is a chemically synthesized RNA molecule. The invention also relates to RNA products produced by the methods and the uses of such products.

Description

[0001] RNA PRODUCTS AND METHODS OF PRODUCTION

[0002] TECHNICAL FIELD

[0003] The invention relates to methods for producing an RNA product (e.g. a guide RNA or prime editing guide RNA (pegRNA)) comprising ligating at least three RNA molecules. The at least three RNA molecules comprise: a first RNA molecule that is a chemically synthesized RNA molecule, a second RNA molecule that is an in vitro transcription (IVT) produced RNA molecule, and a third RNA molecule that is a chemically synthesized RNA molecule. The invention also relates to RNA products produced by the methods and the uses of such products.

[0004] BACKGROUND

[0005] There are a wide range of uses for RNA molecules including research applications, diagnostic applications, RNA therapeutics (such as gene therapies e.g. therapies based on ASO, siRNA and CRISPR-Cas) and mRNA-based vaccines (e.g. mRNA-based COVID-19 vaccines) (Pfeifer et al., 2023, npj Systems Biology and Applications (2023) 9:60, https: / / doi.org / 10.1038 / s41540-023-00323-3).

[0006] RNA molecules may be obtained in three ways: purification from biological sources; chemical synthesis using a solid-phase method; or enzymatic synthesis by in vitro transcription (IVT). All of these approaches have limitations. Purification from cells is suitable for complex or large and highly abundant RNAs; however, it is not ideal for the production of RNA therapeutics (or other applications for which high purity RNA molecules are required). Chemical synthesis allows the generation of high purity RNA molecules with modifications, but its major drawback is the size limitation of about 100 nucleotides. For larger RNA molecules, RNA is typically generated by in vitro transcription (Flemmich et al., 2024, Angew. Chem. Int. Ed. 2024, 63, e202403063). Methods have also emerged to address the production of specific RNAs. For example, Lei et al., have developed such a method that combines two RNA molecules to generate prime editing guide RNA (pegRNA) (Nat Biotechnol (2024). https: / / doi.Org / 10.1038 / S41587-024-02394-x).

[0007] Notwithstanding the availability of this range of techniques, there is a need for methods that enable the production of large and chemically modified RNAs with a high yield and purity. There is also a need for methods that provide these benefits at a reasonable cost.

[0008] DESCRIPTION

[0009] The inventors have developed methods for producing RNA products (including large and chemically modified RNA products). The methods can be used to produce a high yield of an RNA product and the RNA product may have a high purity. Furthermore, the methods enable the production of more stable RNA molecules that are resistant to degradation (e.g. degradation by exonucleases). The methods also enable the production of RNA products in a cost-effective manner for the applications for which they are required.

[0010] 1

[0011] 17228500 RGM1 RGM1The invention provides methods for producing an RNA product (e.g. a guide RNA or prime editing guide RNA (pegRNA)) comprising ligating at least three RNA molecules. The at least three RNA molecules comprise: a first RNA molecule that is a chemically synthesized RNA molecule, a second RNA molecule that is an in vitro transcription (IVT) produced RNA molecule, and a third RNA molecule that is a chemically synthesized RNA molecule. The approach means that RNA products can be designed in a rational way such that: IVT may be used to synthesize a large RNA molecule (herein the second RNA molecule) that does not require chemical modification; and chemical synthesis may be used to synthesize smaller RNA molecules (herein the first and third RNA molecules) that do require chemical modification (e.g. to increase the stability of the RNA product).

[0012] The invention provides methods for producing an RNA product (e.g. a guide RNA or prime editing guide RNA (pegRNA)) comprising ligating at least three RNA molecules by splint ligation. The at least three RNA molecules comprise: a first RNA molecule that is a chemically synthesized RNA molecule, a second RNA molecule that is an in vitro transcription (IVT) produced RNA molecule, and a third RNA molecule that is a chemically synthesized RNA molecule. The approach means that RNA products can be designed in a rational way such that: IVT may be used to synthesize a large RNA molecule (herein the second RNA molecule) that does not require chemical modification; and chemical synthesis may be used to synthesize smaller RNA molecules (herein the first and third RNA molecules) that do require chemical modification (e.g. to increase the stability of the RNA product).

[0013] A method for producing an RNA product, wherein the method comprises:

[0014] a) providing a first RNA molecule, a second RNA molecule and a third RNA molecule; and b) ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule;

[0015] wherein the first RNA molecule is a chemically synthesized RNA molecule, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule, and the third RNA molecule is a chemically synthesized RNA molecule.

[0016] A method for producing an RNA product, wherein the method comprises:

[0017] a) providing a first RNA molecule, a second RNA molecule and a third RNA molecule; and b) ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule, wherein the ligating is performed by splint ligation;

[0018] wherein the first RNA molecule is a chemically synthesized RNA molecule, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule, and the third RNA molecule is a chemically synthesized RNA molecule.

[0019] The first RNA molecule may comprise a nuclease-resistant nucleotide and the third RNA molecule may comprise a nuclease-resistant nucleotide.

[0020] 2

[0021] 17228500 RGM1 RGM1The RNA product may be a linear RNA product.

[0022] The RNA product may comprise a long RNA, a long non-coding RNA (IncRNA), a non-coding RNA (ncRNA), a guide RNA (gRNA), a single guide RNA (sgRNA), a prime editing guide RNA (pegRNA), base-editing guide RNA or an mRNA (e.g. an mRNA encoding a pathogen-specific antigen or an mRNA encoding a neoantigen). The gRNA may comprise an aptamer sequence (e.g. MS2 aptamer sequence).

[0023] The RNA product may comprise an RNA cassette. The RNA cassette may comprise a long RNA, a long non-coding RNA (IncRNA), a non-coding RNA (ncRNA), a guide RNA (gRNA), a prime editing guide RNA (pegRNA), base-editing guide RNA or an mRNA (e.g. an mRNA encoding a pathogenspecific antigen or an mRNA encoding a neoantigen). The gRNA may comprise an aptamer sequence (e.g. MS2 aptamer sequence).

[0024] The RNA product may be at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, at least 105 nucleotides, at least 110 nucleotides, at least 115 nucleotides, at least 120 nucleotides, at least 125 nucleotides, at least 135 nucleotides, at least 145 nucleotides, at least 150 nucleotides, at least 155 nucleotides, at least 160 nucleotides, at least 165 nucleotides, at least 170 nucleotides, at least 175 nucleotides, at least 180 nucleotides, at least 185 nucleotides, at least 190 nucleotides, at least 195 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 750 nucleotides, at least 1000 nucleotides, at least 1500 nucleotides, at least 2000 nucleotides, at least 3000 nucleotides, at least 4000 nucleotides, at least 5000 nucleotides, at least 6000 nucleotides, at least 7000 nucleotides, at least 8000 nucleotides or at least 9000 nucleotides. Preferably, the RNA product is at least 120 nucleotides.

[0025] The RNA product may be a protected linear RNA. The RNA product may comprise two or more nuclease resistant nucleotides (i.e. nucleotides resistant to exonuclease digestion). The RNA product may be resistant to 5’-3’ exonuclease digestion and / or 3’-5’ exonuclease digestion. The RNA product may comprise at least 1 nuclease resistant nucleotide 5’ of the RNA cassette and at least 1 nuclease resistant nucleotide 3’ of the RNA cassette. The RNA product may comprise at least 2 nuclease resistant nucleotides 5’ of the RNA cassette and at least 2 nuclease resistant nucleotides 3’ of the RNA cassette. The RNA product may comprise at least 3 nuclease resistant nucleotides 5’ of the RNA cassette and at least 3 nuclease resistant nucleotides 3’ of the RNA cassette. The RNA product may comprise at least 4 nuclease resistant nucleotides 5’ of the RNA cassette and at least 4 nuclease resistant nucleotides 3’ of the RNA cassette. The RNA product may comprise at least 5 nuclease resistant nucleotides 5’ of the RNA cassette and at least 5 nuclease resistant nucleotides 3’ of the RNA cassette. Preferably, the RNA product comprises at least 2 nuclease resistant nucleotides 5’ of the RNA cassette and at least 2 nuclease resistant nucleotides 3’ of the RNA cassette.

[0026] The invention provides a method for producing an RNA product, wherein the method comprises:

[0027] 3

[0028] 17228500 RGM1 RGM1a) providing a first RNA molecule, a second RNA molecule, and a third RNA molecule,; and b) ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule, wherein the ligating is performed by splint ligation;

[0029] wherein the first RNA molecule is a chemically synthesized RNA molecule, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule, and the third RNA molecule is a chemically synthesized RNA molecule.

[0030] The invention provides for a method for producing an RNA product, wherein the method comprises: a) providing a first RNA molecule, a second RNA molecule, and a third RNA molecule, wherein the first RNA molecule comprises a nuclease-resistant nucleotide and the third RNA molecule comprises a nuclease-resistant nucleotide; and

[0031] b) ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule; wherein the guide RNA comprises, in the 5’-3’ direction, a spacer sequence and a scaffold region; and wherein the first RNA molecule is a chemically synthesized RNA molecule, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule, and the third RNA molecule is a chemically synthesized RNA molecule.

[0032] In the methods, step (b) may comprise: annealing the first, second and third RNA molecules to a splint DNA; and ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule by splint ligation.

[0033] The step of ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule by splint ligation may be performed using T4 RNA ligase 2.

[0034] In the methods, step (b) may comprise: annealing the first, second and third RNA molecules to a splint DNA; and ligating (e.g. using T4 RNA ligase 2) the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating (e.g. using T4 RNA ligase 2) the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule by splint ligation.

[0035] In the methods, step (b) may comprise: ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule to generate an intermediate ligation product; annealing the first RNA molecule and the intermediate ligation product to a splint DNA; and ligating the 3’ end of the first RNA molecule to the 5’ end of the intermediate ligation product.

[0036] In the methods, step (b) may comprise: ligating (e.g. using T4 RNA ligase 1) the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule to generate an intermediate ligation product; annealing the first RNA molecule and the intermediate ligation product to a splint DNA; and ligating (e.g.

[0037] 4

[0038] 17228500 RGM1 RGM1using T4 RNA ligase 2) the 3’ end of the first RNA molecule to the 5’ end of the intermediate ligation product.

[0039] In the methods, step (b) may comprise: ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule to generate an intermediate ligation product; annealing the third RNA molecule and the intermediate ligation product to a splint DNA; and ligating the 5’ end of the third RNA molecule to the 3’ end of the intermediate ligation product.

[0040] In the methods, step (b) may comprise: ligating (e.g. using T4 RNA ligase 1) the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule to generate an intermediate ligation product; annealing the third RNA molecule and the intermediate ligation product to a splint DNA; and ligating (e.g. using T4 RNA ligase 2) the 5’ end of the third RNA molecule to the 3’ end of the intermediate ligation product.

[0041] In the methods, the splint DNA may be a single splint DNA molecule.

[0042] In the methods, step (b) may be performed using T4 RNA ligase.

[0043] In the methods, ligation may be performed using a single-stranded RNA ligase e.g. T4 RNA ligase 1.

[0044] In the methods, ligation may be performed using a double-stranded RNA ligase e.g. T4 RNA ligase 2.

[0045] In the methods, step (b) may comprise: ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule by an RNA ligase.

[0046] The RNA ligase may be T4 RNA ligase 1.

[0047] In the methods, the first RNA may comprise at least a portion of the spacer sequence and the second RNA molecule may comprise at least a portion of the scaffold region.

[0048] In the methods, the guide RNA may be a prime editing guide RNA (pegRNA) and the pegRNA may comprise, in the 5’-3’ direction, a spacer sequence, a scaffold region, a reverse transcription template (RTT) and a primer binding site (PBS).

[0049] In the methods, either:

[0050] a) the second RNA molecule may comprise the reverse transcription template (RTT);

[0051] b) the third RNA molecule may comprise the reverse transcription template (RTT); or c) the second RNA molecule may comprise a portion of the reverse transcription template (RTT) and the third RNA molecule may comprise a portion of the reverse transcription template (RTT).

[0052] 5

[0053] 17228500 RGM1 RGM1The third RNA molecule may comprise the PBS.

[0054] In the methods, step (a) may comprise: producing the first RNA molecule and the third RNA molecule by chemical synthesis; and producing the second RNA molecule by in vitro transcription (IVT), optionally wherein the chemical synthesis comprises solid-phase synthesis.

[0055] In the methods, the splint DNA may be a protected DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the protected DNA comprises x nuclease-resistant nucleotides at the 5’ end of the ssDNA cassette or 5’ of the ssDNA cassette, and y nuclease-resistant nucleotides at the 3’-end of the ssDNA cassette or 3’ of the ssDNA cassette, wherein x is at least 1 and y is at least 1.

[0056] In the methods, the splint DNA may be a protected DNA comprising a single-stranded DNA (ssDNA) cassette, and the method may comprise:

[0057] (a) providing a partially protected double-stranded DNA (dsDNA) comprising a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises a cassette, x nuclease-resistant nucleotides at the 5’ end of the cassette or 5’ of the cassette, and y nuclease-resistant nucleotides at the 3’-end of the cassette or 3’ of the cassette, wherein x is at least 1 and y is at least 1 ; and

[0058] (b) digesting the second strand of the partially protected dsDNA with an exonuclease thereby generating the protected DNA.

[0059] The RNA product may comprise a long RNA, a long non-coding RNA (IncRNA), a non-coding RNA (ncRNA), a guide RNA (gRNA), a single guide RNA (sgRNA), a prime editing guide RNA (pegRNA), base-editing guide RNA or an mRNA (e.g. an mRNA encoding a pathogen-specific antigen or an mRNA encoding a neoantigen). The gRNA may comprise an aptamer sequence (e.g. MS2 aptamer sequence).

[0060] In the methods, the RNA product may comprise a guide RNA (gRNA), such as a single guide RNA (sgRNA), a prime editing guide RNA (pegRNA) or a base-editing guide RNA.

[0061] The invention provides a method for producing a guide RNA, wherein the method comprises:

[0062] a) providing a first RNA molecule, a second RNA molecule, and a third RNA molecule, wherein the first RNA molecule comprises a nuclease-resistant nucleotide and the third RNA molecule comprises a nuclease-resistant nucleotide; and

[0063] b) ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule; wherein the guide RNA comprises, in the 5’-3’ direction, a spacer sequence and a scaffold region; and

[0064] 6

[0065] 17228500 RGM1 RGM1wherein the first RNA molecule is a chemically synthesized RNA molecule, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule, and the third RNA molecule is a chemically synthesized RNA molecule.

[0066] In the methods, step (b) may comprise: annealing the first, second and third RNA molecules to a splint DNA; and ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule by splint ligation.

[0067] The step of ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule by splint ligation may be performed using T4 RNA ligase 2.

[0068] In the methods, step (b) may comprise: annealing the first, second and third RNA molecules to a splint DNA; and ligating (e.g. using T4 RNA ligase 2) the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating (e.g. using T4 RNA ligase 2) the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule by splint ligation.

[0069] In the methods, step (b) may comprise: ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule to generate an intermediate ligation product; annealing the first RNA molecule and the intermediate ligation product to a splint DNA; and ligating the 3’ end of the first RNA molecule to the 5’ end of the intermediate ligation product.

[0070] In the methods, step (b) may comprise: ligating (e.g. using T4 RNA ligase 1) the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule to generate an intermediate ligation product; annealing the first RNA molecule and the intermediate ligation product to a splint DNA; and ligating (e.g. using T4 RNA ligase 2) the 3’ end of the first RNA molecule to the 5’ end of the intermediate ligation product.

[0071] In the methods, step (b) may comprise: ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule to generate an intermediate ligation product; annealing the third RNA molecule and the intermediate ligation product to a splint DNA; and ligating the 5’ end of the third RNA molecule to the 3’ end of the intermediate ligation product.

[0072] In the methods, step (b) may comprise: ligating (e.g. using T4 RNA ligase 1) the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule to generate an intermediate ligation product; annealing the third RNA molecule and the intermediate ligation product to a splint DNA; and ligating (e.g. using T4 RNA ligase 2) the 5’ end of the third RNA molecule to the 3’ end of the intermediate ligation product.

[0073] In the methods, the splint DNA may be a single splint DNA molecule.

[0074] 7

[0075] 17228500 RGM1 RGM1In the methods, step (b) may be performed using T4 RNA ligase.

[0076] In the methods, ligation may be performed using a single-stranded RNA ligase e.g. T4 RNA ligase 1.

[0077] In the methods, ligation may be performed using a double-stranded RNA ligase e.g. T4 RNA ligase 2.

[0078] In the methods, step (b) may comprise: ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule by an RNA ligase.

[0079] The RNA ligase may be T4 RNA ligase 1.

[0080] In the methods, the first RNA may comprise at least a portion of the spacer sequence and the second RNA molecule may comprise at least a portion of the scaffold region.

[0081] In the methods, the guide RNA may be a prime editing guide RNA (pegRNA) and the pegRNA may comprise, in the 5’-3’ direction, a spacer sequence, a scaffold region, a reverse transcription template (RTT) and a primer binding site (PBS).

[0082] In the methods, either:

[0083] d) the second RNA molecule may comprise the reverse transcription template (RTT);

[0084] e) the third RNA molecule may comprise the reverse transcription template (RTT); or f) the second RNA molecule may comprise a portion of the reverse transcription template (RTT) and the third RNA molecule may comprise a portion of the reverse transcription template (RTT).

[0085] The third RNA molecule may comprise the PBS.

[0086] In the methods, step (a) may comprise: producing the first RNA molecule and the third RNA molecule by chemical synthesis; and producing the second RNA molecule by in vitro transcription (IVT), optionally wherein the chemical synthesis comprises solid-phase synthesis.

[0087] In the methods, the splint DNA may be a protected DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the protected DNA comprises x nuclease-resistant nucleotides at the 5’ end of the ssDNA cassette or 5’ of the ssDNA cassette, and y nuclease-resistant nucleotides at the 3’-end of the ssDNA cassette or 3’ of the ssDNA cassette, wherein x is at least 1 and y is at least 1.

[0088] In the methods, the splint DNA may be a protected DNA comprising a single-stranded DNA (ssDNA) cassette, and the method may comprise:

[0089] 8

[0090] 17228500 RGM1 RGM1(a) providing a partially protected double-stranded DNA (dsDNA) comprising a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises a cassette, x nuclease-resistant nucleotides at the 5’ end of the cassette or 5’ of the cassette, and y nuclease-resistant nucleotides at the 3’-end of the cassette or 3’ of the cassette, wherein x is at least 1 and y is at least 1 ; and

[0091] (b) digesting the second strand of the partially protected dsDNA with an exonuclease thereby generating the protected DNA.

[0092] In the methods, the guide RNA (gRNA) may be a single guide RNA (sgRNA), a prime editing guide RNA (pegRNA) or a base-editing guide RNA.

[0093] The invention provides a method for producing a guide RNA, wherein the method comprises:

[0094] a) providing a first RNA molecule, a second RNA molecule, and a third RNA molecule, wherein the first RNA molecule comprises a nuclease-resistant nucleotide and the third RNA molecule comprises a nuclease-resistant nucleotide; and

[0095] b) ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule, wherein the ligation is performed by splint ligation;

[0096] wherein the guide RNA comprises, in the 5’-3’ direction, a spacer sequence and a scaffold region; and

[0097] wherein the first RNA molecule is a chemically synthesized RNA molecule, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule, and the third RNA molecule is a chemically synthesized RNA molecule.

[0098] The first RNA may comprise at least a portion of the spacer sequence and / or the second RNA molecule may comprise at least a portion of the scaffold region.

[0099] The first RNA may comprise at least a portion of the scaffold region.

[0100] The first RNA may comprise at least a portion of the spacer sequence and at least a portion of the scaffold region.

[0101] The spacer region (or targeting sequence) may anneal to a genomic target sequence.

[0102] The scaffold region is capable of binding a CRISPR-associated endonuclease e.g. Cas9 or Cpf1. The scaffold region may comprise a tracrRNA. The scaffold region may comprise a tracrRNA and a linker region connecting the tracrRNA to the crRNA.

[0103] The invention provides a method for producing a guide RNA, wherein the method comprises:

[0104] 9

[0105] 17228500 RGM1 RGM1a) providing a first RNA molecule comprising a spacer sequence and at least a portion of a scaffold region, a second RNA molecule comprising at least a portion of a scaffold region, and a third RNA molecule comprising at least a portion of a scaffold region, wherein the first RNA molecule comprises a nuclease-resistant nucleotide and the third RNA molecule comprises a nuclease- resistant nucleotide; and

[0106] b) ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule, wherein the ligation is performed by splint ligation;

[0107] wherein the guide RNA comprises, in the 5’-3’ direction, a spacer sequence and a scaffold region; and

[0108] wherein the first RNA molecule is a chemically synthesized RNA molecule, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule, and the third RNA molecule is a chemically synthesized RNA molecule.

[0109] The guide RNA may be a prime editing guide RNA (pegRNA) and wherein the pegRNA comprises, in the 5’-3’ direction, a spacer sequence, a scaffold region, a reverse transcription template (RTT) and a primer binding site (PBS).

[0110] The invention provides a method of producing a prime editing guide RNA (pegRNA), wherein the method comprises:

[0111] a) providing a first RNA molecule, a second RNA molecule comprising a scaffold region, and a third RNA molecule, optionally wherein the first RNA molecule comprises a protected nucleotide and the third RNA molecule comprises a protected nucleotide; and

[0112] b) ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule, wherein the ligating is performed by splint ligation;

[0113] wherein the pegRNA comprises, in the 5’-3’ direction, a spacer sequence, a scaffold region, a reverse transcription template (RTT) and a primer binding site (PBS); and

[0114] wherein the first RNA molecule is a chemically synthesized RNA molecule, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule, and the third RNA molecule is a chemically synthesized RNA molecule.

[0115] The first RNA molecule may comprise at least a portion of the scaffold region.

[0116] The second RNA molecule may comprise the reverse transcription template (RTT). The third RNA molecule may comprise the reverse transcription template (RTT). The second RNA molecule may comprise a portion of the reverse transcription template (RTT) and the third RNA molecule may comprise a portion of the reverse transcription template (RTT).

[0117] The third RNA molecule may comprise the primer binding site (PBS).

[0118] 10

[0119] 17228500 RGM1 RGM1The invention provides a method of producing a prime editing guide RNA (pegRNA), wherein the method comprises:

[0120] a) providing a first RNA molecule comprising a spacer sequence, a second RNA molecule comprising at least a portion of a scaffold region, and a third RNA molecule comprising a primer binding site (PBS), optionally wherein the first RNA molecule comprises a protected nucleotide and the third RNA molecule comprises a protected nucleotide; and

[0121] b) ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule, wherein the ligating is performed by splint ligation;

[0122] wherein the pegRNA comprises, in the 5’-3’ direction, a spacer sequence, a scaffold region, a reverse transcription template (RTT) and a primer binding site (PBS); and

[0123] wherein the first RNA molecule is a chemically synthesized RNA molecule, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule, and the third RNA molecule is a chemically synthesized RNA molecule.

[0124] The first RNA molecule may comprise at least a portion of the scaffold region.

[0125] The second RNA molecule may comprise the reverse transcription template (RTT). The third RNA molecule may comprise the reverse transcription template (RTT). The second RNA molecule may comprise a portion of the reverse transcription template (RTT) and the third RNA molecule may comprise a portion of the reverse transcription template (RTT).

[0126] The invention provides a method of producing a prime editing guide RNA (pegRNA), wherein the method comprises:

[0127] a) providing a first RNA molecule comprising a spacer sequence, a second RNA molecule comprising at least a portion of a scaffold region and at least a portion of a reverse transcription template (RTT), and a third RNA molecule comprising at least a portion of a reverse transcription template (RTT) and a primer binding site (PBS), optionally wherein the first RNA molecule comprises a protected nucleotide and the third RNA molecule comprises a protected nucleotide; and

[0128] b) ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule, wherein the ligating is performed by splint ligation;

[0129] wherein the pegRNA comprises, in the 5’-3’ direction, a spacer sequence, a scaffold region, a reverse transcription template (RTT) and a primer binding site (PBS); and

[0130] wherein the first RNA molecule is a chemically synthesized RNA molecule, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule, and the third RNA molecule is a chemically synthesized RNA molecule.

[0131] The first RNA molecule may comprise at least a portion of the scaffold region.

[0132] 11

[0133] 17228500 RGM1 RGM1The invention provides a method of producing a prime editing guide RNA (pegRNA), wherein the method comprises:

[0134] a) providing a first RNA molecule comprising a spacer sequence, a second RNA molecule comprising at least a portion of a scaffold region, and a third RNA molecule comprising a reverse transcription template (RTT) and a primer binding site (PBS), optionally wherein the first RNA molecule comprises a protected nucleotide and the third RNA molecule comprises a protected nucleotide; and

[0135] b) ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule, wherein the ligating is performed by splint ligation;

[0136] wherein the pegRNA comprises, in the 5’-3’ direction, a spacer sequence, a scaffold region, a reverse transcription template (RTT) and a primer binding site (PBS); and

[0137] wherein the first RNA molecule is a chemically synthesized RNA molecule, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule, and the third RNA molecule is a chemically synthesized RNA molecule.

[0138] The first RNA molecule may comprise at least a portion of the scaffold region.

[0139] In the methods, step (a) may comprise producing the first RNA molecule and the third RNA molecule by chemical synthesis. In the methods, step (a) may comprise producing the second RNA molecule by in vitro transcription (IVT). In the methods, step (a) may comprise: producing the first RNA molecule and the third RNA molecule by chemical synthesis; and producing the second RNA molecule by in vitro transcription (IVT).

[0140] The chemical synthesis may comprise solid-phase synthesis (e.g. solid-phase synthesis using phosphoramidite chemistry).

[0141] The step of producing the second RNA by in vitro transcription may comprise producing an adaptor-ligated DNA product as a template for in vitro transcription (IVT). Methods for producing an adaptor-ligated DNA product are described herein.

[0142] The step of producing the second RNA by in vitro transcription may comprise producing a PCR product as a template for in vitro transcription (IVT).

[0143] The step of producing the second RNA by in vitro transcription may comprise using chemical synthesis to produce the template for in vitro transcription (IVT). The chemical synthesis may comprise solidphase synthesis (e.g. solid-phase synthesis using phosphoramidite chemistry).

[0144] 12

[0145] 17228500 RGM1 RGM1In the methods, the second RNA molecule may be produced by IVT in a reaction that comprises GMP. The reaction may comprise a higher concentration of GMP than GTP. The reaction may comprise a ratio of at least 3:1 , at least 4:1 or at least 5:1 (GMP:GTP)

[0146] In the methods, IVT may be performed using at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 15 mM or at least 20 mM NTPs. Preferably, IVT is performed using at least 5 mM NTPs.

[0147] In the methods, IVT may be performed using 3-20 mM, 4-15 mM or 5-10 mM NTPs.

[0148] In the methods, IVT may be performed using a T3 RNA polymerase, a T7 RNA polymerase, an SP6 RNA polymerase, a Therminator polymerase, an E. coli RNA polymerase (Core Enzyme), E. coli RNA polymerase (Holoenzyme) and / or VSW-3 RNA polymerase.

[0149] In the methods, IVT may be performed using any T7 like RNA polymerase or an engineered version of T7 RNA polymerase.

[0150] In the methods, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule. The 5’ end of the second RNA molecule may have a 5’-triphosphate. The 5’ end of the second RNA molecule may have a 5’-terminal triphosphate. Preferably, the 5’ end of the second RNA molecule has a 5’-terminal GTP.

[0151] The in vitro transcription (IVT) produced RNA molecule may be processed prior to step (b) (ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule) to generate a second RNA molecule. The second RNA molecule used in step (b) may have a 5’-monophosphate. The second RNA molecule used in step (b) may have a 5’-terminal monophosphate. The second RNA molecule may have a 5’-terminal GMP or a 5’-terminal AMP. There are described herein various methods for processing the in vitro transcription (IVT) produced RNA molecule prior to step (b).

[0152] In the methods, the second RNA molecule may have a 5’-terminal GTP and step (a) may comprise treating the second RNA molecule with a phosphatase to cleave phosphate groups from the 5’-terminal GTP to produce a second RNA molecule with a 5’-terminal GMP. The phosphatase may be E. coli 5’-RNA pyrophosphohydrolase (RppH). The phosphatase may be an alkaline phosphatase, wherein the alkaline phosphatase removes all phosphate groups from the 5’ GTP, and the step of treating the second RNA with a phosphatase may be followed by treatment with a T4 polynucleotide kinase to phosphorylate guanosine and generate a 5’-terminal GMP.

[0153] In the methods, the second RNA molecule may have a 5’-terminal GTP and step (a) may comprise treating the second RNA molecule with a polyphosphatase to cleave phosphate groups from the 5’-

[0154] 13

[0155] 17228500 RGM1 RGM1terminal GTP to produce a second RNA molecule with a 5’-terminal GMP. In the methods, the second RNA molecule may have a 5’-terminal GTP and step (a) may comprise treating the second RNA molecule with a 5’ pyrophosphohydrolase (e.g. RppH) to cleave phosphate groups from the 5’-terminal GTP to produce a second RNA molecule with a 5’-terminal GMP.

[0156] In the methods, the 5’ end of the second RNA molecule, which has been generated by in vitro transcription (IVT) in the presence of nucleotide triphosphates (NTPs), may have an uncapped 5’ triphosphate end (GTP) that is refractory to enzymatic ligation. Thus, the second RNA molecule may have a 5’-terminal GTP and step (a) may comprise treating the second RNA enzymatically with RNA 5’ phosphatase (e.g. a polyphosphatase) or 5’ pyrophosphohydrolase (e.g. RppH) to cleave phosphate groups from the 5’-terminal GTP to produce a second RNA molecule with a 5’-terminal GMP which is amenable to enzymatic ligation. The second RNA molecule may have a 5’-terminal GTP and step (a) may comprise treating the second RNA enzymatically with alkaline phosphatase (e.g. calf intestinal alkaline phosphatase (CIAP)), wherein the alkaline phosphatase removes all phosphate groups from the 5’ GTP, and the step of treating the second RNA molecule with the alkaline phosphatase may be followed by treatment with a T4 polynucleotide kinase to phosphorylate guanosine and generate a 5’-terminal GMP.

[0157] In the methods, step (a) may comprise cleaving (i.e. digesting) the second RNA molecule (produced by IVT) by a restriction endonuclease (e.g. a endoribonuclease). The restriction endonuclease may cleave the second RNA molecule (produced by IVT) to generate a second RNA molecule prior to ligation of the second RNA molecule in step (b).

[0158] IVT may be performed using T7 RNA polymerase or SP6 like bacteriophage RNA polymerase. These enzymes require a GG sequence in the template used for IVT. In the methods, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule. Thus, the first two nucleotides of the second RNA molecule may be GG. Some template sequences for IVT may not include a GG sequence or may include a GG sequence at an undesired position.

[0159] In the methods, an RNA molecule generated by in vitro transcription may be enzymatically digested by EcoToxNI , a novel site-specific RNA endonuclease which cleaves at the site GAA / AU, to generate the second RNA molecule with a 5’OH AU end. The GAA / AU site is conserved in all gRNA scaffolds. The 5’OH end is converted to a 5’-monophosphate enzymatically via phosphor transfer with a kinase such as T4 polynucleotide kinase in the presence of ATP. In the method, the use of EcoToxNI means that the use of in vitro transcription to generate the second RNA molecule does not depend on the presence of a GG sequence in the guide RNA sequence. The template for IVT can be generated with an additional sequence which includes a GG sequence which is upstream of the sequence of the second RNA molecule. This additional sequence will be cleaved off by EcoToxNI .

[0160] 14

[0161] 17228500 RGM1 RGM1In the methods, step (a) may comprise cleaving (i.e. digesting) the second RNA molecule (produced by IVT) by EcoToxNI . The second RNA molecule (produced by IVT) may have a GAA / AU site. The EcoToxNI may cleave the second RNA molecule (produced by IVT) to generate a second RNA molecule with a 5’OH AU end. The step of cleaving the second RNA with EcoToxNI may be followed by treatment with a T4 polynucleotide kinase to phosphorylate adenosine and generate a 5’-terminal AMP.

[0162] In the methods, step (b) may comprise: annealing the first, second and third RNA molecules to a splint DNA; and ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule. Preferably, the splint DNA is a single splint DNA molecule.

[0163] Preferably, the splint DNA is a single stranded DNA molecule.

[0164] The splint DNA may be two splint DNA molecules. The first RNA molecule may be annealed (completely or partially) to a first splint DNA molecule, the second RNA molecule may be partially annealed to the first splint DNA molecule and partially annealed to a second splint DNA molecule, and the third RNA molecule may be annealed (completely or partially) to the second splint DNA molecule.

[0165] The splint DNA may be a protected DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the protected DNA comprises x nuclease-resistant nucleotides at the 5’ end of the ssDNA cassette or 5’ of the ssDNA cassette, and y nuclease-resistant nucleotides at the 3’-end of the ssDNA cassette or 3’ of the ssDNA cassette, wherein x is at least 1 and y is at least 1.

[0166] The splint DNA may not comprise any nuclease-resistant nucleotides.

[0167] The splint DNA may be a chemically synthesized DNA molecule. The method may comprise producing the splint DNA by chemical synthesis. The chemical synthesis may comprise solid-phase synthesis (e.g. solid-phase synthesis using phosphoramidite chemistry).

[0168] In the methods, the template for in vitro transcription (IVT) and / or the splint DNA may be chemically synthesized DNA molecules. The chemical synthesis may comprise solid-phase synthesis (e.g. solidphase synthesis using phosphoramidite chemistry).

[0169] In the methods, the step of producing the second RNA by in vitro transcription may comprise using chemical synthesis to produce the template for in vitro transcription (IVT) and using chemical synthesis to produce the splint DNA. The chemical synthesis may comprise solid-phase synthesis (e.g. solidphase synthesis using phosphoramidite chemistry).

[0170] The splint DNA may be a protected DNA comprising a single-stranded DNA (ssDNA) cassette, and wherein the method comprises:

[0171] 15

[0172] 17228500 RGM1 RGM1(a) providing a partially protected double-stranded DNA (dsDNA) comprising a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises a cassette, x nuclease-resistant nucleotides at the 5’ end of the cassette or 5’ of the cassette, and y nuclease-resistant nucleotides at the 3’-end of the cassette or 3’ of the cassette, wherein x is at least 1 and y is at least 1 ; and

[0173] (b) digesting the second strand of the partially protected dsDNA with an exonuclease thereby generating the protected DNA.

[0174] The splint DNA may comprise a sequence comprising regions complementary to the scaffold region and spacer sequence. The splint DNA may comprise a sequence comprising regions complementary to the primer binding site (PBS), reverse transcription template (RTT), scaffold region and / or spacer sequence. The splint DNA may anneal to at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the RNA product sequence (e.g. guide RNA or pegRNA sequence).

[0175] The method may further comprise digesting the splint DNA using DNase. The DNase may be DNase I and / or Duplex DNase. Additionally or alternatively, the step of digesting the splint DNA may be performed using one or more exonucleases e.g. Exolll.

[0176] The method may further comprise digesting the unligated RNA molecules using one or more RNA exonucleases. The RNA exonuclease(s) may be a 5’-3’ RNA exonuclease and / or a 3’-5’ RNA exonuclease. The one or more RNA exonucleases may be selected from XRN-I (5’ to 3’), Terminator 5'-Phosphate-Dependent Exonuclease (5’ to 3’), Exonuclease T (3’ to 5’), Exolll (3’ to 5’) and / or RNase R (3’ to 5’).

[0177] In the methods, the step of splint ligation may be performed using T3 DNA ligase, Thermostable 5' App DNA / RNA Ligase, SplintR® Ligase, T4 RNA ligase and / or RtcB Ligase. Preferably the step of splint ligation is performed using T4 RNA ligase. The T4 RNA ligase may be T4 RNA ligase 1 or T4 RNA ligase 2.

[0178] In the methods, when the first, second and third RNA molecules are annealed to a splint DNA, the ligation of the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and the ligation of the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule may be performed using a double-stranded RNA ligase (e.g. T4 RNA ligase 2).

[0179] In the methods, when the first, second and third RNA molecules are annealed to a splint DNA, the ligation of the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and the ligation of the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule may be performed using T4 RNA ligase 2.

[0180] In the methods, when the 3’ end of the second RNA molecule is ligated to the 5’ end of the third RNA molecule to generate an intermediate ligation product, the ligation may be performed using a single-

[0181] 16

[0182] 17228500 RGM1 RGM1stranded RNA ligase (e.g. T4 RNA ligase 1). When the first RNA molecule and the intermediate ligation product are annealed to a splint DNA, the ligation of the 3’ end of the first RNA molecule to the 5’ end of the intermediate ligation product may be performed using a double-stranded RNA ligase (e.g. T4 RNA ligase 2).

[0183] In the methods, when the 3’ end of the second RNA molecule is ligated to the 5’ end of the third RNA molecule to generate an intermediate ligation product, the ligation may be performed using T4 RNA ligase 1. When the first RNA molecule and the intermediate ligation product are annealed to a splint DNA, the ligation of the 3’ end of the first RNA molecule to the 5’ end of the intermediate ligation product may be performed using T4 RNA ligase 2.

[0184] In the methods, when the 3’ end of the first RNA molecule is ligated to the 5’ end of the second RNA molecule to generate an intermediate ligation product, the ligation may be performed using a singlestranded RNA ligase (e.g. T4 RNA ligase 1). When the third RNA molecule and the intermediate ligation product are annealed to a splint DNA, the ligation of the 5’ end of the third RNA molecule to the 3’ end of the intermediate ligation product may be performed using a double-stranded RNA ligase (e.g. T4 RNA ligase 2).

[0185] In the methods, when the 3’ end of the first RNA molecule is ligated to the 5’ end of the second RNA molecule to generate an intermediate ligation product, the ligation may be performed using T4 RNA ligase 1. When the third RNA molecule and the intermediate ligation product are annealed to a splint DNA, the ligation of the 5’ end of the third RNA molecule to the 3’ end of the intermediate ligation product may be performed using T4 RNA ligase 2.

[0186] In the methods, the first RNA molecule may comprise a protected nucleotide 5’ of the spacer sequence and / or in a 5’ end region. The third RNA molecule may comprise a nuclease-resistant nucleotide 3’ of the primer binding site (e.g. if the RNA product is a pegRNA) and / or in a 3’ end region.

[0187] The method may be a cell-free method. The method may be an in vitro method. The method may be a cell-free, in vitro method.

[0188] The invention provides a guide RNA for use in a method of treating a disease, wherein the guide RNA is produced according to the method of any one of claims 1-14 and wherein the guide RNA is used to either (i) edit a target gene by gene editing and thereby treat the disease or (ii) activate the transcription of endogenous gene and thereby treat the disease.

[0189] The invention provides a guide RNA for use in a method of treating a disease, wherein the guide RNA is produced according to any one of the methods described herein and wherein the guide RNA is used to either (i) edit a target gene by gene editing and thereby treat the disease or (ii) activate the transcription of endogenous gene and thereby treat the disease.

[0190] 17

[0191] 17228500 RGM1 RGM1The invention provides a method of treating a disease by gene editing comprising: a) producing a guide RNA according to any of the methods described herein; and b) using the guide RNA to either (i) edit a target gene by gene editing and thereby treat the disease or (ii) activate the transcription of endogenous gene and thereby treat the disease.

[0192] The invention provides a pegRNA for use in a method of treating a disease, wherein the pegRNA is produced according to any of the methods described herein and wherein the pegRNA is used to either (i) edit a target gene by prime editing and thereby treat the disease or (ii) activate the transcription of endogenous gene and thereby treat the disease.

[0193] The invention provides a method of treating a disease by prime editing comprising: a) producing a pegRNA according to any of the methods described herein; and b) using the pegRNA to either (i) edit a target gene by prime editing and thereby treat the disease or (ii) activate the transcription of endogenous gene and thereby treat the disease.

[0194] The invention provides a method of producing an RNA vaccine, wherein the method comprises:

[0195] (a) producing an RNA product using any of the methods described herein, wherein the RNA product comprises an mRNA encoding a pathogen-specific antigen; and

[0196] (b) formulating the RNA product to produce the RNA vaccine.

[0197] The invention provides a method of producing an RNA vaccine, wherein the method comprises:

[0198] (a) producing an RNA product using any of the methods described herein, wherein the RNA product comprises an mRNA encoding a neoantigen; and

[0199] (b) formulating the RNA product to produce the RNA vaccine.

[0200] 1. Properties of the first, second and third RNA molecules

[0201] The first RNA molecule and / or the third RNA molecule may (each) comprise at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides or at least 110 nucleotides. Preferably, the first RNA molecule and / or the third RNA molecule (each) comprise at least 10 nucleotides.

[0202] The first RNA molecule and / or the third RNA molecule may (each) comprise less than 100 nucleotides, less than 90 nucleotides, less than 80 nucleotides, less than 70 nucleotides, less than 60 nucleotides, less than 50 nucleotides, less than 45 nucleotides, less than 40 nucleotides, less than 35 nucleotides, less than 30 nucleotides, less than 25 nucleotides, less than 20 nucleotides, less than 15 nucleotides, less than 10 nucleotides or less than 5 nucleotides. Preferably, the first RNA molecule and / or the third RNA molecule may (each) comprise less than 25 nucleotides.

[0203] 18

[0204] 17228500 RGM1 RGM1The first RNA molecule and / or the third RNA molecule may (each) comprise 5-120 nucleotides, 10-110 nucleotides, 15-100 nucleotides, 20-90 nucleotides, 30-80 nucleotides, 40-70 nucleotides or 50-60 nucleotides. Preferably, the first RNA molecule and / or the third RNA molecule (each) comprise 10-100 nucleotides.

[0205] The second RNA molecule may comprise at least 50, at least 75 nucleotides, at least 100, at least 105 nucleotides, at least 110 nucleotides, at least 115 nucleotides, at least 120 nucleotides, at least 125 nucleotides, at least 135 nucleotides, at least 145 nucleotides at least 150, at least 155 nucleotides, at least 160 nucleotides, at least 165 nucleotides, at least 170 nucleotides, at least 175 nucleotides, at least 180 nucleotides, at least 185 nucleotides, at least 190 nucleotides, at least 195 nucleotides, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 325, at least 350, at least 375, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, or at least 9000 nucleotides. Preferably, the second RNA molecule comprises at least 105 nucleotides.

[0206] The first RNA molecule may encode a spacer sequence. The second RNA molecule may encode a scaffold region and a first portion of a MS2 stem loop sequence. The third RNA molecule may encode a second portion of the MS2 stem loop sequence.

[0207] The first RNA molecule may encode a spacer sequence, the second RNA molecule may encode a scaffold region and a first portion of a MS2 stem loop sequence, and the third RNA molecule may encode a second portion of the MS2 stem loop sequence.

[0208] 2. Methods for producing an adaptor-ligated DNA product as a template for IVT

[0209] The methods may comprise producing an adaptor-ligated DNA product as a template for in vitro transcription (IVT) to produce the second RNA molecule.

[0210] The methods may comprise producing an adaptor-ligated DNA product, wherein the method comprises: (a) contacting an amplified DNA product with an endonuclease and first and second adaptor molecules to form a single contiguous aqueous volume; and

[0211] (b) incubating the single contiguous aqueous volume to generate an adaptor-ligated DNA product, wherein the adaptor-ligated DNA product comprises a linear double-stranded region, and wherein the first adaptor molecule is appended to a first end of the linear double-stranded region and the second adaptor molecule is appended to a second end of the linear double-stranded region.

[0212] Preferably, the step of contacting the amplified DNA product with the endonuclease, the ligase and first and second adaptor molecules is performed in a single reaction (i.e. a single step).

[0213] 19

[0214] 17228500 RGM1 RGM1The first and second adaptor molecules may be identical molecules or they may be different molecules. For example, the first adaptor molecule and / or the second adaptor molecule may comprise a hairpin. The first adaptor molecule and / or the second adaptor molecule may be double-stranded linear nucleic acid molecules comprising one or more nuclease-resistant nucleotides. The first adaptor molecule may comprise a hairpin and the second adaptor molecule may be a double-stranded linear nucleic acid molecule comprising one or more nuclease-resistant nucleotides. Thus, the adaptor-ligated DNA product may be resistant to nuclease (e.g. exonuclease) digestion.

[0215] The step of contacting the amplified DNA product with the endonuclease and first and second adaptor molecules is preferably performed in the presence of a ligase. Thus, the invention provides a method for producing an adaptor-ligated DNA product, wherein the method comprises:

[0216] (a) contacting the amplified DNA product with an endonuclease, a ligase and first and second adaptor molecules to form a single contiguous aqueous volume; and

[0217] (b) incubating the single contiguous aqueous volume to generate the adaptor-ligated DNA product, wherein the adaptor-ligated DNA product comprises a linear double-stranded region, and wherein the first adaptor molecule is appended to a first end of the linear double-stranded region and the second adaptor molecule is appended to a second end of the linear double-stranded region.

[0218] The appending (or linking or closing) of the first adaptor molecule and / or the second adaptor molecule may be performed by hybridization or ligation of the adaptor molecules to the ends of the digested DNA product. The digested DNA product may comprise or consist of a linear double-stranded region. Thus, the first adaptor molecule may be hybridized to the first end of the linear double-stranded region. The second adaptor molecule may be hybridized to the second end of the linear double-stranded region. The first adaptor molecule may be ligated to the first end of the linear double-stranded region. The second adaptor molecule may be ligated to the second end of the linear double-stranded region. The appending of the first adaptor molecule and the second adaptor molecule may be performed by both hybridization and ligation of the adaptor molecules to the ends of the linear double-stranded region. Thus, the first adaptor molecule may be hybridized and ligated to the first end of the linear doublestranded region. The second adaptor molecule may be hybridized and ligated to the second end of the linear double-stranded region. The appending may be performed via a linker or spacer molecule which facilitates joining of the adaptor molecule to the first and / or second end of the linear double-stranded region.

[0219] The linker or spacer may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, or at least 200 base pairs long.

[0220] The methods may comprise producing an adaptor-ligated DNA product, wherein the method comprises: (a) amplifying a DNA template molecule as described herein, wherein the DNA template molecule comprises at least one endonuclease target sequence to generate amplified DNA product;

[0221] 20

[0222] 17228500 RGM1 RGM1(b) contacting the amplified DNA product with an endonuclease, a ligase and first and second adaptor molecules to form a single contiguous aqueous volume; and

[0223] (c) incubating the single contiguous aqueous volume to generate the adaptor-ligated DNA product, wherein the adaptor-ligated DNA product comprises a linear double-stranded region, and wherein the first adaptor molecule is appended to a first end of the linear double-stranded region and the second adaptor molecule is appended to a second end of the linear double-stranded region.

[0224] The methods may further comprise, after the step of amplification and before the step of contacting the amplified DNA product with an endonuclease, a ligase and first and second adaptor molecules, a step of heat-deactivation. Thus, the invention provides a method for producing an adaptor-ligated DNA product, the method comprises:

[0225] (a) amplifying a DNA template molecule as described herein, wherein the DNA template molecule comprises at least one endonuclease target sequence to generate amplified DNA product;

[0226] (b) heat-deactivation of the reaction of step (a);

[0227] (c) contacting the amplified DNA product with an endonuclease, a ligase and first and second adaptor molecules to form a single contiguous aqueous volume; and

[0228] (d) incubating the single contiguous aqueous volume to generate the adaptor-ligated DNA product, wherein the adaptor-ligated DNA product comprises a linear double-stranded region, and wherein the first adaptor molecule is appended to a first end of the linear double-stranded region and the second adaptor molecule is appended to a second end of the linear double-stranded region.

[0229] The step of heat-deactivation may be performed under conditions sufficient to inactive the reagents used during the amplification reaction. The step of heat-deactivation may be performed at a temperature of at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C, or at least 100°C. The step of heat-deactivation may be performed for at least 1 min, at least 3 mins, at least 5 mins, at least 10 mins, at least 15 mins, or at least 20 mins.

[0230] In the method described herein, after the step of amplification, the step of contacting the amplified DNA product with an endonuclease, a ligase and first and second adaptor molecules to form a single contiguous aqueous volume may be performed without purifying the product of the amplification reaction. That is to say that the step of contacting the amplified DNA product with an endonuclease, a ligase and first and second adaptor molecules may be performed directly after the step of amplification. The step of contacting the amplified DNA product with an endonuclease, a ligase and first and second adaptor molecules may be performed directly after the step of heat-deactivation.

[0231] The method may further comprise, after the step of incubating the single contiguous aqueous volume, a step of purification of the adaptor-ligated DNA product.

[0232] 21

[0233] 17228500 RGM1 RGM1The method may further comprise, after the step of incubating the single contiguous aqueous volume, a step of nuclease digestion. The nuclease digestion may be exonuclease digestion, such as exonuclease I and / or exonuclease III digestion. The step of nuclease digestion may take place before or after the step of purification. The step of nuclease digestion may allow for removal of any doublestranded DNA molecules and / or adaptor molecules which have not been used to produce adaptor-ligated DNA product. Thus, the method for producing an adaptor-ligated DNA product may comprise the steps:

[0234] (a) contacting the amplified DNA product with an endonuclease, a ligase and first and second adaptor molecules to form a single contiguous aqueous volume;

[0235] (b) incubating the single contiguous aqueous volume to generate the adaptor-ligated DNA product, wherein the adaptor-ligated DNA product comprises a linear double-stranded region, and wherein the first adaptor molecule is appended to a first end of the linear double-stranded region and the second adaptor molecule is appended to a second end of the linear double-stranded region; and

[0236] (c) incubating the single contiguous aqueous volume with a nuclease (e.g. an exonuclease).

[0237] The method for producing an adaptor-ligated DNA product may comprise the steps:

[0238] (a) amplifying a DNA template molecule as described herein, wherein the DNA template molecule comprises at least one endonuclease target sequence to generate amplified DNA product;

[0239] (b) contacting the amplified DNA product with an endonuclease, a ligase and first and second adaptor molecules to form a single contiguous aqueous volume;

[0240] (c) incubating the single contiguous aqueous volume to generate the adaptor-ligated DNA product, wherein the adaptor-ligated DNA product comprises a linear double-stranded region, and wherein the first adaptor molecule is appended to a first end of the linear double-stranded region and the second adaptor molecule is appended to a second end of the linear double-stranded region; and

[0241] (d) incubating the single contiguous aqueous volume with a nuclease (e.g. an exonuclease).

[0242] The method for producing an adaptor-ligated DNA product may comprise the steps:

[0243] (a) amplifying a DNA template molecule as described herein, wherein the DNA template molecule comprises at least one endonuclease target sequence;

[0244] (b) contacting the amplified DNA product with an endonuclease, a ligase and first and second adaptor molecules to form a single contiguous aqueous volume;

[0245] (c) incubating the single contiguous aqueous volume to generate the adaptor-ligated DNA product, wherein the adaptor-ligated DNA product comprises a linear double-stranded region, and wherein the first adaptor molecule is appended to a first end of the linear double-stranded region and the second adaptor molecule is appended to a second end of the linear double-stranded region;

[0246] (d) purification of the adaptor-ligated DNA product; and

[0247] (e) incubating the product of step (d) with a nuclease (e.g. an exonuclease).

[0248] The method for producing an adaptor-ligated DNA product may comprise the steps:

[0249] 22

[0250] 17228500 RGM1 RGM1(a) amplifying a DNA template molecule as described herein, wherein the DNA template molecule comprises at least one endonuclease target sequence;

[0251] (b) contacting the amplified DNA product with an endonuclease, a ligase and first and second adaptor molecules to form a single contiguous aqueous volume;

[0252] (c) incubating the single contiguous aqueous volume to generate the adaptor-ligated DNA product, wherein the adaptor-ligated DNA product comprises a linear double-stranded region, and wherein the first adaptor molecule is appended to a first end of the linear double-stranded region and the second adaptor molecule is appended to a second end of the linear double-stranded region;

[0253] (d) incubating the single contiguous aqueous volume with a nuclease (e.g. an exonuclease); and (e) purification of the DNA product.

[0254] The endonuclease may be a restriction endonuclease (such as a Type II restriction endonuclease), RNA-guided DNA endonuclease, meganuclease or homing endonuclease. The Type II restriction endonuclease may be a Type IIP restriction endonuclease (e.g. EcoRI, Hindlll, BamHI or Notl) or a Type IIS restriction endonuclease (e.g. Bsal, Bbvl, Fokl or Sapl). The RNA-guided DNA endonuclease may be an endonuclease of Class 2 e.g. Class 2 Type V. The RNA-guided DNA endonuclease may be Cas12a. The RNA-guided DNA endonuclease may be Cpf1 or Mad7. Preferably, the RNA-guided DNA endonuclease is Cpf1. The homing endonuclease may be l-Ceul, l-Scel, Pl-Pspl or Pl-Scel.

[0255] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a Type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside of the recognition sequence. For example, the endonuclease may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsell, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstVIl, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104l, Earl, Ecil, Eco31l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp11091, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI restriction enzyme.

[0256] Type IIS restriction endonucleases cleave the amplified DNA molecule outside of the recognition sequence (i.e. an endonuclease target sequence), which means that the recognition sequence (i.e. endonuclease target sequence) is not included in the adaptor-ligated DNA product.

[0257] The step of incubating the single contiguous aqueous volume may be performed under conditions that promote appending (or linking) of the first and second adaptor molecules to the digested DNA product. The appending may be performed by creating a covalent link between the first and / or second adaptor molecule and the first and / or second end of the digested DNA product.

[0258] 23

[0259] 17228500 RGM1 RGM1P262744WQ00

[0260] The step of incubating the single contiguous aqueous volume may be performed under conditions that promote digestion of the amplified DNA product to produce the digested DNA product. The digestion of the amplified DNA product to produce the digested DNA product may be performed at a first temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or at about 37°C. The digestion may be endonuclease digestion, preferably Type IIS endonuclease digestion.

[0261] The step of incubating the single contiguous aqueous volume may be performed under conditions that promote ligation of the digested DNA products to the first and second adaptor molecules. The ligation may be at least 5%, at least 10%, at least 15, at least 20%, at least 25%, at least 30%, at least 35%, at least 40, at least 45%, at least 50%, at least 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% efficient. For example, at least 5%, at least 10%, at least 15, at least 20%, at least 25%, at least 30%, at least 35%, at least 40, at least 45%, at least 50%, at least 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the digested DNA products may be incorporated into adaptor-ligated DNA products. Preferably, the ligation is at least 15% efficient.

[0262] Ligation efficiency may be established based on DNA quantification values before and after the digestion / ligation reaction. Thus, ligation efficiency may be established based on the equation: (starting amplified DNA amount) I (final linear DNA amount) x 100%.

[0263] Ligation efficiency may also be established based on DNA quantification values before and after the digestion / ligation reaction and the subsequent exonuclease treatment to remove remaining DNA constructs and adaptor molecules excess.

[0264] For example, the amplified DNA molecule generated by amplification (e.g. rolling circle amplification) may first be quantified so that the amount of the starting material during the digestion / ligation reaction is known. After all the enzymatic reactions, the adaptor-ligated DNA product may be quantified to calculate the ligation efficiency as per the equation above.

[0265] DNA quantification methods are known to a person skilled in the art. For example, DNA quantifications may be carried out using the Qubit dsDNA BR assay from ThermoFisher (https: / / www.thermofisher.eom / order / catalog / product / Q32850# / Q32850).

[0266] The step of ligation of the digested DNA product to the first and second adaptor molecules may be performed at a second temperature of 1°C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 11°C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C.

[0267] The step of incubating the single contiguous aqueous volume may comprise incubating at a first temperature and then incubating at a second temperature. The first temperature may be 1°C-100°C,

[0268] 24

[0269] 17228500 RGM1 RGM11°C-80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C-45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or about 37°C. The second temperature may be 1°C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 11°C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C. Preferably, the first temperature is 35°C-39°C and the second temperature is 14°C-18°C. Using these conditions the endonuclease may be a Type IIS restriction endonuclease (e.g. Bsal) and the ligase may be T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase.

[0270] The step of incubating the single contiguous aqueous volume may be performed isothermally. The step of incubating the single contiguous aqueous volume may comprise incubating at a constant temperature. The constant temperature promotes simultaneous digestion of the amplified DNA product to produce the digested DNA product and ligation of the digested DNA product to the first and second adaptor molecules. For example, the constant temperature may be 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. Preferably, the constant temperature is 30°C. The constant temperature is intended to mean that the temperature does not significantly change during the reaction. The constant temperature is intended to mean that the temperature variation during the step of incubating the single contiguous aqueous volume is less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1°C. In a preferred embodiment the temperature during the step of incubating the single contiguous aqueous does not deviate by more than 5°C, preferably by not more than 3°C, even more preferably not more than 1 °C. Thus, the constant temperature may be a temperature in a range of 20°C-30°C, 22°C-32°C, 24°C-34°C, 26°C-36°C, 28°C-38°C, 30°C-40°C, 22°C-28°C, 32°C-38°C, 25°C-35°C, 26°C-34°C, 27°C- 33°C, 27.5°C-32.5°C, 28°C-32°C, 28.5°C-31 ,5°C, 29°C-31 °C, or 29.5°C-30.5°C. Preferably, the constant temperature is a temperature in a range of 27.5°C-32.5°C. Alternatively, the constant temperature may be a temperature in a range of 32°C-42°C, 33°C-41 °C, 34°C-40°C, 35°C-39°C, 36°C-38°C. Preferably, the constant temperature is a temperature in a range of 34.5°C-39.5°C.

[0271] The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 80, at least 90, or at least 100 times, preferably at least 20 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature less than 40, less than 35, less than 30 times, less than 29, less than 25 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature 2-100, 5-80, 10-70, 20-60, or 30-60 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature 2-20, 5-29, 61-100, or 65-80 times.

[0272] 25

[0273] 17228500 RGM1 RGM1P262744WQ00

[0274] The DNA template molecule may comprise at least one endonuclease target sequence. Preferably, the DNA template molecule comprises at least two endonuclease target sequences. The endonuclease target sequences may be the same or different. Preferably, the at least one endonuclease target sequence is a restriction endonuclease target sequence. Different restriction endonuclease target sequences would be known to the skilled person. The endonuclease target sequence may be a Type IIS restriction endonuclease target sequence. For example, the restriction endonuclease target sequence may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsell, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV11, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104l, Earl, Ecil, Eco31l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp11091, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequence. The at least one endonuclease target sequence may be a native endonuclease target sequence (i.e. an endonuclease target sequence present in the template molecule). Alternatively, the at least one endonuclease target sequence may be introduced to the DNA template molecule prior to the production of the adaptor-ligated DNA product.

[0275] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a Type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside of the recognition sequence. For example, the endonuclease may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsell, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstVIl, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104l, Earl, Ecil, Eco31l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp11091, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI restriction enzyme.

[0276] The ligase may be a DNA ligase, such as a T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase.

[0277] The first adaptor molecule and / or the second adaptor molecule may comprise one or more locked nucleic acids (LNAs).

[0278] The first adaptor molecule and / or the second adaptor molecule may comprise one or more protected nucleotides (i.e. nuclease-resistant nucleotides), such as phosphorothioated nucleotides.

[0279] 26

[0280] 17228500 RGM1 RGM1The first and second adaptor molecules may comprise one or more phosphorothioated nucleotides, such that, once the adaptor molecules are appended (e.g. ligated) to the digested DNA product, the adaptor-ligated DNA product is resistant to nuclease digestion or has improved or enhanced resistance to nuclease digestion. The adaptor-ligated DNA product may be resistant to 3’-end exonuclease digestion (e.g. by exonuclease III) and / or 5’-end exonuclease digestion (e.g. by exonuclease VIII).

[0281] The adaptor molecule may comprise a plurality of phosphorothioated nucleotides. For example, the adaptor molecules may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15 or at least 16 phosphorothioated nucleotides in each strand.

[0282] The adaptor molecule may be a nucleic acid adaptor molecule. The adaptor molecule may be doublestranded. The adaptor molecule may comprise a portion that is double-stranded.

[0283] The first and / or second adaptor molecules may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, or at least 16 base pairs.

[0284] The adaptor molecule may comprise a plurality of phosphorothioated nucleotides in each strand. For example, the adaptor molecules may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15 or at least 16 phosphorothioated nucleotides in each strand.

[0285] The adaptor molecule may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the adaptor molecules may comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15 or at least 16 phosphorothioated nucleotides at internal positions in each strand. Preferably, the adaptor molecule comprises at least 2 phosphorothioated nucleotides at internal positions in each strand.

[0286] The internal positions may not be located between the second and penultimate nucleotide of the adaptor molecule. The internal positions may be any position in the adaptor molecules other than the last nucleotide at the end of each strand.

[0287] The adaptor molecule may comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of protected nucleotides.

[0288] The nucleotides resistant to exonuclease digestion (i.e. protected nucleotides) may be phosphorothioated nucleotides of at least one type. For example, the at least one type of

[0289] 27

[0290] 17228500 RGM1 RGM1phosphorothioated nucleotides is a-S-dATP (i.e. 2’-deoxyadenosine-5’-(a-thio)-triphosphate), a-S-dCTP (i.e. 2’-deoxycytidine-5’-(a-thio)-triphosphate), a-S-dGTP (i.e. 2’-deoxyguanosine-5’-(a-thio)-triphosphate), a-S-dTTP (i.e. 2’-deoxythymidine-5’-(a-thio)-triphosphate), a-S-dUTP (i.e. 2’-deoxyuridine-5’-(a-thio)-triphosphate), and / or uridine 2’, 3’-cyclophosphorothioate.

[0291] The adaptor molecules may comprise at least two types of phosphorothioated nucleotides. For example, the at least two types of phosphorothioated nucleotides are: a-S-dATP and a-S-dCTP, a-S-dATP and a-S-dGTP, a-S-dATP and a-S-dTTP, a-S-dCTP and a-S-dGTP, a-S-dCTP and a-S-dTTP, or a-S-dGTP and a-S-dTTP.

[0292] The adaptor molecules may comprise at least three types of phosphorothioated nucleotides. For example, the at least three types of phosphorothioated nucleotides are:

[0293] a. a-S-dATP, a-S-dCTP and a-S-dGTP;

[0294] b. a-S-dATP, a-S-dCTP and a-S-dTTP;

[0295] c. a-S-dATP, a-S-dGTP and a-S-dTTP; or

[0296] d. a-S-dCTP, a-S-dGTP and a-S-dTTP.

[0297] The adaptor molecules may comprise at least four types of phosphorothioated nucleotides. For example, the at least four types of protected nucleotides are a-S-dATP, a-S-dCTP, a-S-dGTP and a-S-dTTP.

[0298] The phosphorothioated nucleotides may be Sp-isomers, Rp-isomers or a mixture of both Sp- and Rp-isomers.

[0299] The nucleotides resistant to exonuclease digestion (i.e. protected nucleotides) may be MOE nucleotides of at least one type, or at least two, three or four types. For example, the MOE nucleotides may be 2’-O-methoxy-ethyl guanosine, 2’-O-methoxy-ethyl cytidine, 2’-O-methoxy-ethyl adenosine, and / or 2’-O-methoxy-ethyl thymidine.

[0300] The first end of the digested DNA product may be complementary to a portion of the first adaptor molecule. The second end of the digested DNA product may be complementary to a portion of the second adaptor molecule. The first end and / or the second end of the digested DNA product may be generated by endonuclease digestion as described herein.

[0301] As used herein, the term “complementary” refers to the pairing of nucleotide sequences according to Watson / Crick pairing rules. For example, a sequence 5’-GCGGTCCCA-3’ has the complementary sequence of 5’-TGGGACCGC-3’. A complement sequence can also be a sequence of RNA complementary to the DNA sequence.

[0302] 28

[0303] 17228500 RGM1 RGM1The first adaptor molecule and / or the second adaptor molecule may confer resistance to the nuclease digestion, such as exonuclease digestion (e.g. exonuclease I and / or exonuclease III digestion).

[0304] The first adaptor molecule may be a nucleic acid adaptor molecule. The second adaptor molecule may be a nucleic acid adaptor molecule. The first adaptor molecule and / or the second adaptor molecule may comprise a self-complementary element which creates a loop, such as a hairpin loop or a stem loop. Thus, the first adaptor molecule may comprise a hairpin or a stem-loop. The second adaptor molecule may comprise a hairpin or a stem-loop. Both the first and second adaptor molecules may comprise a hairpin or a stem-loop. The adaptor molecules may each comprise a double-stranded portion comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are linked together in a hairpin such that the sense strand is hybridized to the antisense strand. The double-stranded portion of an adaptor may comprise a 3’ overhang or a 5’ overhang of at least 1 , at least 2, at least 3, at least 4, or at least 5 nucleotides. Preferably the 3’ overhang or the 5’ overhang is 4-8 nucleotides. Each end of the linear double-stranded region (or linear portion of the doublestranded DNA molecule) may comprise a 3’ or a 5’ overhang. A portion of the first adaptor molecule (e.g. the overhang) may be complementary to the first end of the linear double-stranded region. A portion of the second adaptor molecule may be complementary to the second end of the linear doublestranded region.

[0305] The adaptor-ligated DNA product may be an open linear DNA product, optionally wherein the linear DNA product comprises a first adaptor molecule at a first end and a second adaptor molecule at a second end. The adaptor-ligated DNA product may be a partially closed linear DNA, optionally wherein the linear double-stranded region is closed at a first end by a first adaptor molecule or closed at a second end by a second adaptor molecule. A partially closed linear DNA product may result from one of the first or second adaptor molecules being a linear double-stranded DNA molecule and other comprising a hairpin or stem-loop. The adaptor-ligated DNA product may be closed linear DNA product, optionally wherein the linear double-stranded region is closed at a first end by a first adaptor molecule and closed at a second end by a second adaptor molecule. A closed linear DNA product may result from both the first and second adaptor molecules comprising a hairpin or stem-loop. Preferably the adaptor-ligated DNA product is a partially closed linear DNA product.

[0306] Linear double-stranded adaptors may comprise nuclease-resistant nucleotides as described herein thereby conferring exonuclease resistance on the adaptor-ligated DNA product. Methods for producing closed linear DNA products, linear double-stranded DNA products and partially closed linear DNA products are provided in W02023 / 006978A1 , which is incorporated herein by reference.

[0307] 3. Protected splint DNA and methods for producing a protected splint DNA

[0308] The protected splint DNA may have enhanced resistance to exonuclease digestion. The enhanced resistance to exonuclease digestion may extend the life of the ssDNA cassette in a cell-free system (i.e. the ssDNA cassette may have enhanced resistance to extracellular exonucleases). The ssDNA

[0309] 29

[0310] 17228500 RGM1 RGM1cassette may be protected from digestion by exonucleases that cleave the 3’-end nucleotides (e.g. exonuclease I), exonucleases that cleave the 5’-end nucleotides (e.g. exonuclease VIII) and exonucleases that cleave both the 3’ and 5’ end nucleotides (e.g. exonuclease VII).

[0311] The protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise a nuclease-resistant nucleotide at the 5’ end of the ssDNA cassette or 5’ of the ssDNA cassette, and a nuclease-resistant nucleotides at the 3’-end of the ssDNA cassette or 3’ of the ssDNA cassette, wherein the cassette comprises at least 100 nucleotides. The protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise at least 3 nuclease-resistant nucleotides at the 5’ end of the ssDNA cassette or 5’ of the ssDNA cassette, and at least 3 nuclease-resistant nucleotides at the 3’-end of the ssDNA cassette or 3’ of the ssDNA cassette, optionally wherein the ssDNA cassette comprises at least 100 nucleotides. The protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise at least 5 nuclease-resistant nucleotides at the 5’ end of the ssDNA cassette or 5’ of the ssDNA cassette, and at least 5 nuclease-resistant nucleotides at the 3’-end of the ssDNA cassette or 3’ of the ssDNA cassette, optionally wherein the ssDNA cassette comprises at least 100 nucleotides. The protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise x nuclease-resistant nucleotides at the 5’ end of the ssDNA cassette or 5’ of the ssDNA cassette, and y nuclease-resistant nucleotides at the 3’-end of the ssDNA cassette or 3’ of the ssDNA cassette, wherein x is at least 1 and y is at least 1 , optionally wherein the ssDNA cassette comprises at least 100 nucleotides.

[0312] x and y may be independently selected from at least 1 , at least 2, at least 3, at least 4 and at least 5. x and y may be different or the same. Preferably x and y are both at least 3. x and y may both be at least 5. x may be 3 and y may be 1 and vice versa, x may be 3 and y may be 5 and vice versa.

[0313] “Protected splint DNA” as used herein refers to the protected splint DNA comprising a single-stranded DNA (ssDNA) cassette unless specified otherwise.

[0314] The protected splint DNA may be resistant to 3’-end exonuclease digestion (e.g. by exonuclease III) and / or 5’-end exonuclease digestion (e.g. by exonuclease VIII). The protected splint DNA may be resistant to 3’-end exonuclease digestion (e.g. by exonuclease III, exonuclease T and / or exonuclease VII) and / or 5’-end exonuclease digestion (e.g. by exonuclease VIII, exonuclease VII, RecJf, RecJ, T7 exonuclease, Lambda exonuclease and / or T5 exonuclease).

[0315] The protected splint DNA comprises nuclease-resistant nucleotides (i.e. protected nucleotides), such as phosphorothioated nucleotides.

[0316] The protected DNA may be a single-stranded DNA molecule i.e. the protected DNA may not comprise any double-stranded regions.

[0317] 30

[0318] 17228500 RGM1 RGM1The protected splint DNA may comprise at least 50, at least 75, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 250, at least 300, at least 400, at least 500, at least 750, at least 1000 nucleotides, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, or at least 9000 nucleotides. Preferably, the protected splint DNA comprises at least 135 nucleotides.

[0319] The ssDNA cassette may comprise at least 25, at least 50, at least 75, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125 nucleotides, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 250, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, or at least 9000 nucleotides. Preferably, the ssDNA cassette comprises at least 125 nucleotides.

[0320] The ssDNA cassette may be a single cassette. The term “single cassette” as used herein is intended to encompass a product that does not comprise or consist of a plurality of cassettes. That is to say that the protected DNA comprises only a single cassette, which may comprise a single sequence complementary to a pegRNA. The single cassette may not comprise or consist of a plurality of tandem repeat sequences, and / or concatemeric DNA. The term “single cassette” as used herein is intended to encompass a single copy of the DNA sequence of interest, for example, a single copy of the coding sequence. Thus, the “single cassette” may not encompass a cassette that comprises or consist of multiple copies of the same DNA sequence linked in series.

[0321] The ssDNA cassette comprises sequences complementary to the sequences of the first RNA molecule, the second RNA molecule and the third RNA molecule.

[0322] The protected splint DNA may additionally comprise a plurality of protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions. For example, the protected splint DNA may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, or at least 250 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions. Preferably, the protected splint DNA comprises at least 2 protected nucleotides (e.g. phosphorothioated nucleotides) at internal positions.

[0323] The internal positions may not be located between the second and penultimate nucleotide of the protected splint DNA.

[0324] 31

[0325] 17228500 RGM1 RGM1The splint DNA may be a protected splint DNA and the methods may comprise producing the protected splint sDNA comprising a single-stranded DNA (ssDNA) cassette (e.g. a long ssDNA cassette). The method may comprise producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the protected splint DNA comprises a nuclease-resistant nucleotide at the 5’ end of the ssDNA cassette or 5’ of the ssDNA cassette, and a nuclease-resistant nucleotide at the 3’-end of the ssDNA cassette or 3’ of the ssDNA cassette. The nuclease-resistant nucleotides are preferably exonuclease-resistant nucleotides e.g. phosphorothioated nucleotides. The location of exonucleaseresistant nucleotides at the 5’ and 3’ ends of and / or outside of the ssDNA cassette protects the ssDNA cassette from exonuclease digestion. For example, it provides protection from digestion by exonucleases that cleave the 3’-end nucleotides (e.g. exonuclease III) and exonucleases that cleave the 5’-end nucleotides (e.g. exonuclease VIII).

[0326] The enhanced resistance to exonuclease digestion extends the life of the ssDNA cassette in a cell-free system (i.e. the ssDNA cassette has enhanced resistance to extracellular exonucleases).

[0327] The methods may comprise producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the method comprises:

[0328] (a) providing a partially protected double-stranded DNA (dsDNA) comprising a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises a cassette, a nuclease-resistant nucleotide at the 5’ end of the cassette or 5’ of the cassette, and a nuclease-resistant nucleotide at the 3’-end of the cassette or 3’ of the cassette; and

[0329] (b) digesting the second strand of the partially protected dsDNA with an exonuclease (or an exonuclease mixture) thereby generating the protected splint DNA.

[0330] The invention provides a method for producing a protected DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the method comprises:

[0331] (a) providing a partially protected double-stranded DNA (dsDNA) comprising a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises a cassette, at least 3 nuclease-resistant nucleotides at the 5’ end of the cassette or 5’ of the cassette, and at least 3 nuclease-resistant nucleotides at the 3’-end of the cassette or 3’ of the cassette; and

[0332] (b) digesting the second strand of the partially protected dsDNA with an exonuclease (or an exonuclease mixture) thereby generating the protected DNA.

[0333] The invention provides a method for producing a protected DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the method comprises:

[0334] (a) providing a partially protected double-stranded DNA (dsDNA) comprising a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises a cassette, at least 5 nuclease-resistant nucleotides at the 5’ end of the cassette or 5’ of the cassette, and at least 5 nuclease-resistant nucleotides at the 3’-end of the cassette or 3’ of the cassette; and

[0335] 32

[0336] 17228500 RGM1 RGM1(b) digesting the second strand of the partially protected dsDNA with an exonuclease (or an exonuclease mixture) thereby generating the protected DNA.

[0337] The splint DNA may be a protected DNA comprising a single-stranded DNA (ssDNA) cassette, and wherein the method comprises:

[0338] (a) providing a partially protected double-stranded DNA (dsDNA) comprising a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises a cassette, x nuclease-resistant nucleotides at the 5’ end of the cassette or 5’ of the cassette, and y nuclease-resistant nucleotides at the 3’-end of the cassette or 3’ of the cassette, wherein x is at least 1 and y is at least 1 ; and

[0339] (b) digesting the second strand of the partially protected dsDNA with an exonuclease (or an exonuclease mixture) thereby generating the protected DNA.

[0340] The exonuclease (or exonuclease mixture) may comprise at least a 5’ to 3’ exonuclease and / or a 3’ to 5’ exonuclease. The exonuclease mixture may comprise at least three exonucleases, wherein at least one of the at least three exonucleases is a 5’ to 3’ exonuclease and at least one of the at least three exonucleases is a 3’ to 5’ exonuclease. The exonuclease mixture may comprise at least three exonucleases, wherein at least two of the at least three exonucleases are 5’ to 3’ exonucleases and at least one of the at least three exonucleases is a 3’ to 5’ exonuclease. The exonuclease mixture may comprise at least three exonucleases, wherein at least one of the at least three exonucleases is a 5’ to 3’ exonuclease and at least two of the at least three exonucleases are 3’ to 5’ exonucleases. The exonuclease mixture may comprise at least four exonucleases, wherein at least one of the at least four exonucleases is a 5’ to 3’ exonuclease and at least one of the at least four exonucleases is a 3’ to 5’ exonuclease. The exonuclease mixture may comprise at least four exonucleases, wherein at least two of the at least four exonucleases are 5’ to 3’ exonucleases and at least one of the at least four exonucleases is a 3’ to 5’ exonuclease. The exonuclease mixture may comprise at least four exonucleases, wherein at least one of the at least four exonucleases is a 5’ to 3’ exonuclease and at least two of the at least four exonucleases are 3’ to 5’ exonucleases. Preferably, the exonuclease mixture comprises at least four exonucleases, wherein at least two of the at least four exonucleases are 5’ to 3’ exonucleases and at least two of the at least four exonucleases are 3’ to 5’ exonucleases.

[0341] The 5’ to 3’ exonuclease(s) may be Lambda exonuclease and / or T7 exonuclease. The 3’ to 5’ exonuclease(s) may be exonuclease I and / or exonuclease III. The 5’ to 3’ exonuclease(s) may be exonuclease VIII.

[0342] The exonuclease mixture may comprise at least two exonucleases selected from a group of exonuclease I, exonuclease III, T7 exonuclease, and Lambda exonuclease. The exonuclease mixture may comprise at least three exonucleases selected from a group of exonuclease I, exonuclease III, T7 exonuclease, and Lambda exonuclease. The exonuclease mixture may comprise exonuclease I, exonuclease III, T7 exonuclease, and Lambda exonuclease.

[0343] 33

[0344] 17228500 RGM1 RGM1The exonuclease mixture may comprise one exonuclease selected from Lambda exonuclease and / or T7 exonuclease, and one exonuclease selected from exonuclease I and / or exonuclease III. For example, the exonuclease mixture may comprise Lambda exonuclease and exonuclease I. For example, the exonuclease mixture may comprise Lambda exonuclease and exonuclease III. For example, the exonuclease mixture may comprise T7 exonuclease and exonuclease I. For example, the exonuclease mixture may comprise T7 exonuclease and exonuclease III.

[0345] The exonuclease mixture may comprise Lambda exonuclease, T7 exonuclease and exonuclease I. The exonuclease mixture may comprise Lambda exonuclease, T7 exonuclease and exonuclease III. The exonuclease mixture may comprise Lambda exonuclease, exonuclease I and exonuclease III. The exonuclease mixture may comprise T7 exonuclease, exonuclease I and exonuclease III.

[0346] The exonuclease mixture may further comprise exonucleases which are able to cleave DNA in both 3’ to 5’ and 5’ to 3’ directions. For example, the exonuclease mixture may comprise exonuclease V.

[0347] The exonucleases from the exonuclease mixture may be added sequentially or simultaneously. Preferably the exonucleases from the exonuclease mixture are added simultaneously.

[0348] x and y may be independently selected from at least 1 , at least 2, at least 3, at least 4 and at least 5. x and y may be different or the same, x and y may both be at least 3. x and y may both be at least 5. x may be 3 and y be 1 and vice versa, x may be 3 and y be 5 and vice versa.

[0349] The partially protected dsDNA may be generated by digesting a precursor dsDNA with an endonuclease and ligating first and second adaptor molecules to the digested precursor dsDNA. Preferably, ligation is performed using T7 DNA ligase. The first and second adaptor molecules may comprise exonucleaseresistant nucleotides e.g. phosphorothioated nucleotides.

[0350] The methods may comprise producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the method comprises:

[0351] (a) generating a partially protected double-stranded DNA (dsDNA) comprising a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises a cassette, x nuclease-resistant nucleotides at the 5’ end of the cassette or 5’ of the cassette, and y nuclease-resistant nucleotides at the 3’-end of the cassette or 3’ of the cassette, wherein x is at least 1 and y is at least 1, wherein the step of generating the partially protected dsDNA comprises:

[0352] (i) contacting a precursor dsDNA comprising a first strand and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0353] 34

[0354] 17228500 RGM1 RGM1(ii) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0355] (iii) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises x nuclease resistant nucleotides and the second adaptor molecule comprises y nuclease-resistant nucleotides and wherein x is at least 1 and y is at least 1 , and wherein the ligase is T7 DNA ligase; and

[0356] (iv) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating the partially protected dsDNA; and

[0357] (b) digesting the second strand of the partially protected dsDNA with an exonuclease mixture thereby generating the protected splint DNA, wherein the exonuclease mixture comprises at least a 5’ to 3’ exonuclease and a 3’ to 5’ exonuclease.

[0358] The methods may comprise producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the method comprises:

[0359] (a) providing a partially protected double-stranded DNA (dsDNA) comprising a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises a cassette, x nuclease-resistant nucleotides at the 5’ end of the cassette or 5’ of the cassette, and y nuclease-resistant nucleotides at the 3’-end of the cassette or 3’ of the cassette, wherein x is at least 1 and y is at least 1 ; and

[0360] (b) digesting the second strand of the partially protected dsDNA with an exonuclease mixture thereby generating the protected splint DNA, wherein the exonuclease mixture comprises at least three exonucleases, wherein at least one of the at least three exonucleases is a 5’ to 3’ exonuclease and at least one of the at least three exonucleases is a 3’ to 5’ exonuclease.

[0361] Importantly, the methods enable the high fidelity and / or high yield production of protected splint DNA comprising long and stable ssDNA cassettes. The methods also enable the production of protected splint DNA of high purity. Methods for producing protected DNA that may be used as protected splint DNA are described in WO2024 / 017978A1 and EP24383218.5, which are incorporated herein by reference.

[0362] The method may be a cell-free method. The method may enable the production of a product comprising the protected DNA that is substantively free of bacterial contaminants (e.g. remaining after cell lysis).

[0363] The partially protected double-stranded DNA (dsDNA) may be any partially protected dsDNA defined herein.

[0364] Digesting the second strand of the partially protected dsDNA with an exonuclease (or exonuclease mixture) may comprise contacting the partially protected dsDNA with an exonuclease (or exonuclease mixture) to digest the second strand.

[0365] 35

[0366] 17228500 RGM1 RGM1Digesting the second strand of the partially protected dsDNA with an exonuclease (or exonuclease mixture) may comprise (a) denaturing the partially protected dsDNA to separate the first and second strands and (b) contacting the second strand with the exonuclease mixture. The partially protected dsDNA may be denatured by means known in the art such as heat and / or alkali conditions.

[0367] Heat may include heating the partially protected dsDNA to a temperature of at least 40°C, at least 50°C, at least 60°C, at least 70°C, at least 80°C, or at least 90°C. Preferably, the partially protected dsDNA is heated to at least 95°C. The heating may be carried out for at least 1 , at least 3, at least 5, at least 10, at least 15, at least 30 or at least 60 minutes. Preferably, the heating is carried out for at least 3 minutes. Thus, the heating may be at 95°C for at least 3 minutes.

[0368] The denatured partially protected dsDNA may be cooled (e.g. to below 20°C, below 10°C or below 5°C) to prevent annealing of the first and second strands. Preferably, the denatured partially protected dsDNA is cooled to around 4°C.

[0369] Alkali conditions may be achieved by adding an alkali reagent (e.g. KOH) that creates a pH above 7, 8, 9, 10, 11 , 12, 13 or 14. Preferably, an alkali reagent is added that creates a pH above 10. After denaturation, the pH may be lowered by adding an acidic reagent (e.g. HCI). The pH may be lowered by an amount which provides conditions for the exonuclease (or exonuclease mixture) to be effective at digesting the second strand.

[0370] If the partially protected dsDNA is denatured, the exonuclease (or exonuclease mixture) may comprise an exonuclease which acts on single-stranded DNA (e.g. E. coli exonuclease I, exonuclease VII, exonuclease T, RecJf, RecJ).

[0371] The step of exonuclease digestion may allow for the removal of any unprotected strands, strands lacking adaptors at both ends and / or remaining adaptor molecules.

[0372] The partially protected dsDNA may be generated by:

[0373] (a) contacting a precursor dsDNA comprising a first strand and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0374] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0375] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises a nuclease resistant nucleotide and the second adaptor molecule comprises a nuclease-resistant nucleotide; and

[0376] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating the partially protected dsDNA.

[0377] 36

[0378] 17228500 RGM1 RGM1Preferably, the ligase is T7 DNA ligase.

[0379] The partially protected dsDNA may be generated by:

[0380] (a) contacting a precursor dsDNA comprising a first strand and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0381] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0382] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises at least 3 nuclease resistant nucleotides and the second adaptor molecule comprises at least 3 nuclease-resistant nucleotides; and

[0383] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating the partially protected dsDNA.

[0384] The partially protected dsDNA may be generated by:

[0385] (a) contacting a precursor dsDNA comprising a first strand and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0386] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0387] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises at least 5 nuclease resistant nucleotides and the second adaptor molecule comprises at least 5 nuclease-resistant nucleotides; and

[0388] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating the partially protected dsDNA.

[0389] The partially protected dsDNA may be generated by:

[0390] (a) contacting a precursor dsDNA comprising a first strand and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0391] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0392] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises x nuclease resistant nucleotides and the second adaptor molecule comprises y nuclease-resistant nucleotides and wherein x is at least 1 and y is at least 1 ; and

[0393] 37

[0394] 17228500 RGM1 RGM1(d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating the partially protected dsDNA.

[0395] The method for producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise:

[0396] (a) contacting a precursor dsDNA comprising a first and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0397] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0398] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first and second adaptor molecules each comprise a nuclease-resistant nucleotide;

[0399] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises the cassette, a nuclease-resistant nucleotide (from the first adaptor molecule) 5’ of the cassette, and a nuclease-resistant nucleotide (from the second adaptor molecule) 3’ of the cassette;

[0400] (e) contacting the partially protected dsDNA with an exonuclease mixture; and

[0401] (f) digesting the second strand of the partially protected dsDNA with the exonuclease mixture thereby generating the protected splint DNA.

[0402] The method for producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise:

[0403] (a) contacting a precursor dsDNA comprising a first and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0404] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0405] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first and second adaptor molecules each comprise at least 3 nuclease-resistant nucleotides;

[0406] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises the cassette, at least 3 nuclease-resistant nucleotides (from the first adaptor molecule) 5’ of the cassette, and at least 3 nuclease-resistant nucleotides (from the second adaptor molecule) 3’ of the cassette;

[0407] 38

[0408] 17228500 RGM1 RGM1(e) contacting the partially protected dsDNA with an exonuclease mixture; and (f) digesting the second strand of the partially protected dsDNA with the exonuclease mixture thereby generating the protected splint DNA.

[0409] Preferably, the ligase is T7 DNA ligase.

[0410] The method for producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise:

[0411] (a) contacting a precursor dsDNA comprising a first and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0412] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0413] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first and second adaptor molecules each comprise at least 5 nuclease-resistant nucleotides;

[0414] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises the cassette, at least 5 nuclease-resistant nucleotides (from the first adaptor molecule) 5’ of the cassette, and at least 5 nuclease-resistant nucleotides (from the second adaptor molecule) 3’ of the cassette;

[0415] (e) contacting the partially protected dsDNA with an exonuclease (or exonuclease mixture); and (f) digesting the second strand of the partially protected dsDNA with the exonuclease (or exonuclease mixture) thereby generating the protected splint DNA.

[0416] The method for producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise:

[0417] (a) contacting a precursor dsDNA comprising a first and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0418] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0419] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises x nuclease-resistant nucleotides and the second adaptor molecule comprises y nuclease-resistant nucleotides, and wherein x is at least 1 and y is at least 1 ;

[0420] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second

[0421] 39

[0422] 17228500 RGM1 RGM1strand, wherein the first strand of the partially protected dsDNA comprises the cassette, x nuclease-resistant nucleotides (from the first adaptor molecule) 5’ of the cassette, and y nuclease-resistant nucleotides (from the second adaptor molecule) 3’ of the cassette;

[0423] (e) contacting the partially protected dsDNA with an exonuclease (or exonuclease mixture); and (f) digesting the second strand of the partially protected dsDNA with the exonuclease (or exonuclease mixture) thereby generating the protected splint DNA.

[0424] Steps (a) (i.e. contacting a precursor dsDNA comprising a first and a second strand with an endonuclease) to (d) (i.e. ligating the first and second adaptor molecules to the digested precursor dsDNA) may be performed in a single contiguous aqueous volume.

[0425] The precursor dsDNA may comprise one or more endonuclease target sequences. The precursor dsDNA may comprise two endonuclease target sequences. The one or more endonuclease target sequences may be Type IIS endonuclease target sequences. The one or more endonuclease target sequences may be Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsell, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV11, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104l, Earl, Ecil, Eco31l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp11091, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequences.

[0426] The method for producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise:

[0427] (a) contacting a precursor dsDNA comprising a first and a second strand with a Type IIS endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, a Type IIS endonuclease target sequence 5’ of the cassette and a Type IIS endonuclease target sequence 3’ of the cassette;

[0428] (b) digesting the precursor dsDNA with the Type IIS endonuclease to generate a digested precursor dsDNA;

[0429] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises x nuclease-resistant nucleotides and the second adaptor molecule comprises y nuclease-resistant nucleotides, and wherein x is at least 1 and y is at least 1 ;

[0430] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises the cassette, x nuclease-

[0431] 40

[0432] 17228500 RGM1 RGM1resistant nucleotides (from the first adaptor molecule) 5’ of the cassette, and y nuclease-resistant nucleotides (from the second adaptor molecule) 3’ of the cassette;

[0433] (e) contacting the partially protected dsDNA with an exonuclease (or exonuclease mixture); and (f) digesting the second strand of the partially protected dsDNA with the exonuclease (or exonuclease mixture) thereby generating the protected splint DNA; optionally wherein steps (a) (i.e. contacting a precursor dsDNA comprising a first and a second strand with a Type IIS endonuclease) to (d) (i.e. ligating the first and second adaptor molecules to the digested precursor dsDNA) are performed in a single contiguous aqueous volume.

[0434] The precursor dsDNA may be a product of amplification. Preferably, the amplification is rolling circle amplification.

[0435] The method may further comprise, before step (a) (i.e. the step of contacting a precursor dsDNA with the endonuclease), a step of amplifying a DNA template to produce the precursor DNA, wherein the DNA template comprises the cassette and the endonuclease target sequences, optionally wherein the DNA template is amplified by rolling circle amplification.

[0436] Thus, the method for producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise:

[0437] (a) amplifying a DNA template to generate a precursor dsDNA, wherein the DNA template comprises a cassette and an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette, optionally wherein the DNA template molecule is amplified by rolling circle amplification;

[0438] (b) contacting the precursor dsDNA comprising a first and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand the cassette, the endonuclease target sequence 5’ of the cassette and the endonuclease target sequence 3’ of the cassette;

[0439] (c) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0440] (d) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first and second adaptor molecules each comprise a nuclease-resistant nucleotide;

[0441] (e) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises the cassette, a nuclease-resistant nucleotide (from the first adaptor molecule) 5’ of the cassette, and a nuclease-resistant nucleotide (from the second adaptor molecule) 3’ of the cassette;

[0442] (f) contacting the partially protected dsDNA with an exonuclease (or exonuclease mixture); and

[0443] 41

[0444] 17228500 RGM1 RGM1(g) digesting the second strand of the partially protected dsDNA with the exonuclease (or exonuclease mixture) thereby generating the protected splint DNA.

[0445] The method for producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise:

[0446] (a) amplifying a DNA template to generate a precursor dsDNA, wherein the DNA template comprises a cassette and an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette, optionally wherein the DNA template molecule is amplified by rolling circle amplification;

[0447] (b) contacting the precursor dsDNA comprising a first and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand the cassette, the endonuclease target sequence 5’ of the cassette and the endonuclease target sequence 3’ of the cassette;

[0448] (c) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0449] (d) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first and second adaptor molecules each comprise at least 3 nuclease-resistant nucleotides;

[0450] (e) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises the cassette, at least 3 nuclease-resistant nucleotides (from the first adaptor molecule) 5’ of the cassette, and at least 3 nuclease-resistant nucleotides (from the second adaptor molecule) 3’ of the cassette;

[0451] (f) contacting the partially protected dsDNA with an exonuclease (or exonuclease mixture); and (g) digesting the second strand of the partially protected dsDNA with the exonuclease (or exonuclease mixture) thereby generating the protected splint DNA.

[0452] The method for producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise:

[0453] (a) amplifying a DNA template to generate a precursor dsDNA, wherein the DNA template comprises a cassette and an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette, optionally wherein the DNA template molecule is amplified by rolling circle amplification;

[0454] (b) contacting the precursor dsDNA comprising a first and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand the cassette, the endonuclease target sequence 5’ of the cassette and the endonuclease target sequence 3’ of the cassette;

[0455] (c) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0456] 42

[0457] 17228500 RGM1 RGM1(d) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first and second adaptor molecules each comprise at least 5 nuclease-resistant nucleotides;

[0458] (e) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises the cassette, at least 5 nuclease-resistant nucleotides (from the first adaptor molecule) 5’ of the cassette, and at least 5 nuclease-resistant nucleotides (from the second adaptor molecule) 3’ of the cassette;

[0459] (f) contacting the partially protected dsDNA with an exonuclease (or exonuclease mixture); and (g) digesting the second strand of the partially protected dsDNA with the exonuclease (or exonuclease mixture) thereby generating the protected splint DNA.

[0460] The method for producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise:

[0461] (a) amplifying a DNA template to generate a precursor dsDNA, wherein the DNA template comprises a cassette and an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette, optionally wherein the DNA template molecule is amplified by rolling circle amplification;

[0462] (b) contacting the precursor dsDNA comprising a first and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand the cassette, the endonuclease target sequence 5’ of the cassette and the endonuclease target sequence 3’ of the cassette;

[0463] (c) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0464] (d) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises x nuclease-resistant and wherein the second adaptor molecule comprises y nuclease-resistant nucleotides, and wherein x is at least 1 and y is at least 1 ;

[0465] (e) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises the cassette, x nuclease-resistant nucleotides (from the first adaptor molecule) 5’ of the cassette, and y nuclease-resistant nucleotides (from the second adaptor molecule) 3’ of the cassette;

[0466] (f) contacting the partially protected dsDNA with an exonuclease (or exonuclease mixture); and (g) digesting the second strand of the partially protected dsDNA with the exonuclease (or exonuclease mixture) thereby generating the protected splint DNA.

[0467] 43

[0468] 17228500 RGM1 RGM1x and y may be independently selected from at least 1 , at least 2, at least 3, at least 4 and at least 5. x and y may be different or the same. Preferably x and y are both at least 3. x and y may both be at least 5. x may be 3 and y be 1 and vice versa, x may be 3 and y be 5 and vice versa.

[0469] Steps (b) (i.e. contacting the precursor dsDNA comprising a first and a second strand with an endonuclease) to (e) (i.e. ligating the first and second adaptor molecules to the digested precursor dsDNA) may be performed in a single contiguous aqueous volume.

[0470] The step of amplifying a DNA template may be an in vitro or in vivo amplification. Preferably, the amplification is an in vitro amplification. For example, the amplification may be performed by rolling circle amplification (RCA), MALBAC method, traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicasedependent amplification (HDA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). Preferably, the amplification is rolling circle amplification.

[0471] Rolling circle amplification may be performed without any primers, or in the presence of a primer or multiple primers. For example, the primer may be a synthetic primer. The primers may be random primers. Rolling circle amplification may be performed in the presence of a primase. The primase may be TthPrimPol. Preferably, if the rolling circle amplification is performed without any primers, it is performed in the presence of a primase, such as TthPrimPol. Similarly, if a primer is used during amplification reaction, a primase is not used. The double-stranded DNA product may be generated by the rolling circle amplification in vitro under isothermal conditions using a suitable nucleic acid polymerase, such as Phi29 DNA polymerase.

[0472] The method may further comprise a step of purification of the digested precursor dsDNA after digesting the precursor dsDNA.

[0473] The method may further comprise a step of purification of the partially protected dsDNA after ligating the first and second adaptor molecules to the digested precursor dsDNA.

[0474] The method may further comprise a step of purification of the protected DNA after digesting the second strand of the partially protected dsDNA with an exonuclease (or exonuclease mixture).

[0475] The method for producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise:

[0476] (a) amplifying a DNA template to generate a precursor dsDNA, wherein the DNA template comprises a cassette and a Type IIS endonuclease target sequence 5’ of the cassette and a Type IIS endonuclease target sequence 3’ of the cassette, wherein the DNA template molecule is amplified by rolling circle amplification;

[0477] 44

[0478] 17228500 RGM1 RGM1(b) contacting the precursor dsDNA comprising a first and a second strand with a Type IIS endonuclease, wherein the precursor dsDNA comprises on the first strand the cassette, the Type IIS endonuclease target sequence 5’ of the cassette and the Type IIS endonuclease target sequence 3’ of the cassette;

[0479] (c) digesting the precursor dsDNA with the Type IIS endonuclease to generate a digested precursor dsDNA;

[0480] (d) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises x nuclease-resistant and wherein the second adaptor molecule comprises y nuclease-resistant nucleotides, and wherein x is at least 1 and y is at least 1 ;

[0481] (e) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises the cassette, x nuclease-resistant nucleotides (from the first adaptor molecule) 5’ of the cassette, and y nuclease-resistant nucleotides (from the second adaptor molecule) 3’ of the cassette;

[0482] (f) contacting the partially protected dsDNA with an exonuclease (or exonuclease mixture); and (g) digesting the second strand of the partially protected dsDNA with the exonuclease (or exonuclease mixture) thereby generating the protected splint DNA; optionally wherein steps (b) (i.e. contacting the precursor dsDNA comprising a first and a second strand with an endonuclease) to (e) (i.e. ligating the first and second adaptor molecules to the digested precursor dsDNA) are performed in a single contiguous aqueous volume.

[0483] The partially protected dsDNA may be generated by:

[0484] (a) contacting a precursor dsDNA comprising a first strand and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0485] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0486] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises a nuclease resistant nucleotide and the second adaptor molecule comprises a nuclease-resistant nucleotide and the first and / or second adaptor molecule comprises an excisable nucleotide; and

[0487] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating the partially protected dsDNA.

[0488] The partially protected dsDNA may be generated by:

[0489] 45

[0490] 17228500 RGM1 RGM1(a) contacting a precursor dsDNA comprising a first strand and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0491] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0492] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises at least 3 nuclease resistant nucleotides and the second adaptor molecule comprises at least 3 nuclease-resistant nucleotides and the first and / or second adaptor molecule comprises an excisable nucleotide or an abasic site; and

[0493] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating the partially protected dsDNA.

[0494] The partially protected dsDNA may be generated by:

[0495] (a) contacting a precursor dsDNA comprising a first strand and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0496] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0497] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises at least 5 nuclease resistant nucleotides and the second adaptor molecule comprises at least 5 nuclease-resistant nucleotides and the first and / or second adaptor molecule comprises an excisable nucleotide or an abasic site; and

[0498] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating the partially protected dsDNA.

[0499] The partially protected dsDNA may be generated by:

[0500] (a) contacting a precursor dsDNA comprising a first strand and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0501] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0502] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises x nuclease resistant nucleotides and the second adaptor molecule comprises y nuclease-resistant nucleotides and wherein x is at least 1 and y is at least 1 and the first and / or second adaptor molecule comprises n excisable nucleotides wherein n is at least 1 ; and

[0503] 46

[0504] 17228500 RGM1 RGM1(d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating the partially protected dsDNA.

[0505] The methods may comprise producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the method comprises:

[0506] (a) contacting a precursor dsDNA comprising a first and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0507] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0508] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first and second adaptor molecules each comprise a nuclease-resistant nucleotide and the first and / or second adaptor molecule comprises an excisable nucleotide;

[0509] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises the cassette, a nuclease-resistant nucleotide (from the first adaptor molecule) 5’ of the cassette, and a nuclease-resistant nucleotide (from the second adaptor molecule) 3’ of the cassette and the second strand comprises an excisable nucleotide;

[0510] (e) contacting the partially protected dsDNA with a DNA glycosylase and an exonuclease (or exonuclease mixture); and

[0511] (f) incubating the partially protected dsDNA with the DNA glycosylase and the exonuclease (or exonuclease mixture) thereby generating the protected splint DNA.

[0512] The method for producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise:

[0513] (a) contacting a precursor dsDNA comprising a first and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0514] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0515] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first and second adaptor molecules each comprise at least 3 nuclease-resistant nucleotides and the first and / or second adaptor molecule comprises an excisable nucleotide;

[0516] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises the cassette, at least 3

[0517] 47

[0518] 17228500 RGM1 RGM1nuclease-resistant nucleotides (from the first adaptor molecule) 5’ of the cassette, and at least 3 nuclease-resistant nucleotides (from the second adaptor molecule) 3’ of the cassette and wherein the second strand comprises an excisable nucleotide (from the first and / or second adaptor molecule);

[0519] (e) contacting the partially protected dsDNA with a DNA glycosylase and an exonuclease (or exonuclease mixture); and

[0520] (f) incubating the partially protected dsDNA with the DNA glycosylase and the exonuclease (or exonuclease mixture) thereby generating the protected splint DNA.

[0521] The method for producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette may comprise:

[0522] (a) contacting a precursor dsDNA comprising a first and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0523] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0524] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first and second adaptor molecules each comprise at least 5 nuclease-resistant nucleotides and the first and / or second adaptor molecule comprises an excisable nucleotide;

[0525] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises the cassette, at least 5 nuclease-resistant nucleotides (from the first adaptor molecule) 5’ of the cassette, and at least 5 nuclease-resistant nucleotides (from the second adaptor molecule) 3’ of the cassette and the second strand comprises at least 2 excisable nucleotides (from the first and / or second adaptor molecule);

[0526] (e) contacting the partially protected dsDNA with a DNA glycosylase and an exonuclease (or exonuclease mixture); and

[0527] (f) incubating the partially protected dsDNA with the DNA glycosylase and the exonuclease (or exonuclease mixture) thereby generating the protected splint DNA.

[0528] The methods may comprise producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the method comprises:

[0529] (a) contacting a precursor dsDNA comprising a first and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0530] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0531] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises x nuclease-resistant nucleotides and n excisable nucleotides and the second adaptor molecule comprises y nuclease-resistant nucleotides

[0532] 48

[0533] 17228500 RGM1 RGM1and m excisable nucleotides, and wherein x is at least 1 and y is at least 1 , wherein n is 0 or at least 1 and m is 0 or at least 1 and n+m is at least 1 ;

[0534] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises the cassette, x nuclease-resistant nucleotides (from the first adaptor molecule) 5’ of the cassette, and y nuclease-resistant nucleotides (from the second adaptor molecule) 3’ of the cassette and the second strand comprises n+m excisable nucleotides;

[0535] (e) contacting the partially protected dsDNA with a DNA glycosylase and an exonuclease (or exonuclease mixture); and

[0536] (f) incubating the partially protected dsDNA with the DNA glycosylase and the exonuclease (or exonuclease mixture) thereby generating the protected splint DNA.

[0537] The partially protected dsDNA may be generated by:

[0538] (a) contacting a precursor dsDNA comprising a first strand and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0539] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0540] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises x nuclease resistant nucleotides and the second adaptor molecule comprises y nuclease-resistant nucleotides and wherein x is at least 1 and y is at least 1 and the first and / or second adaptor molecule comprises n abasic sites wherein n is at least 1 ; and

[0541] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating the partially protected dsDNA.

[0542] The methods may comprise producing a protected splint DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the method comprises:

[0543] (a) contacting a precursor dsDNA comprising a first and a second strand with an endonuclease, wherein the precursor dsDNA comprises on the first strand a cassette, an endonuclease target sequence 5’ of the cassette and an endonuclease target sequence 3’ of the cassette;

[0544] (b) digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA;

[0545] (c) contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, wherein the first adaptor molecule comprises x nuclease-resistant nucleotides and n abasic sites and the second adaptor molecule comprises y nuclease-resistant nucleotides and m abasic sites,

[0546] 49

[0547] 17228500 RGM1 RGM1and wherein x is at least 1 and y is at least 1 , wherein n is 0 or at least 1 and m is 0 or at least 1 and n+m is at least 1 ;

[0548] (d) ligating the first adaptor molecule to a first end of the digested precursor dsDNA and ligating the second adaptor molecule to a second end of the digested precursor dsDNA thereby generating a partially protected dsDNA, wherein the partially protected dsDNA comprises a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises the cassette, x nuclease-resistant nucleotides (from the first adaptor molecule) 5’ of the cassette, and y nuclease-resistant nucleotides (from the second adaptor molecule) 3’ of the cassette and the second strand comprises n+m abasic sites (from the first and / or second adaptor molecules);

[0549] (e) contacting the partially protected dsDNA with an exonuclease (or exonuclease mixture) and optionally with an AP endonuclease; and

[0550] (f) incubating the partially protected dsDNA with the exonuclease (or exonuclease mixture) and the optional AP endonuclease thereby generating the protected splint DNA.

[0551] The endonuclease may be a restriction enzyme endonuclease. The endonuclease may be a Type IIS restriction enzyme. The endonuclease may be any enzyme that recognizes a DNA sequence and cleaves outside of the recognition sequence. For example, the endonuclease may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsell, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstVIl, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104l, Earl, Ecil, Eco31l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp11091, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI restriction enzyme.

[0552] Type IIS restriction endonucleases cleave dsDNA outside of the recognition sequence (i.e. an endonuclease target sequence), which means that the recognition sequence (i.e. endonuclease target sequence) is not included in the cleaved product.

[0553] The ligase may be a DNA ligase, such as a T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase. The ligase is preferably T7 DNA ligase.

[0554] The DNA glycosylase may be OGG1 , MPG, SMUG1 , UNG1 , MBD4, TDG, MYH1 , NTHL1 , NEIL1, NEIL2 or NEIL3.

[0555] The exonuclease may be an exonuclease which acts on single-stranded DNA (e.g. E. coli exonuclease I, exonuclease VII, exonuclease T, RecJf, RecJ). This may be the case when the partially protected dsDNA is denatured.

[0556] 50

[0557] 17228500 RGM1 RGM1Alternatively or in combination, the exonuclease may be an exonuclease which acts on double-stranded DNA (e.g. E. coli exonuclease III, T7 exonuclease, exonuclease V, exonuclease VIII, T5 exonuclease, lambda exonuclease). This may be the case when the partially protected dsDNA is not denatured.

[0558] The protected DNA may be resistant to 3’-end exonuclease digestion (e.g. by exonuclease III) and / or 5’-end exonuclease digestion (e.g. by exonuclease VIII). The protected DNA may be resistant to 3’-end exonuclease digestion (e.g. by exonuclease III, exonuclease I, exonuclease T, exonuclease V and / or exonuclease VII) and / or 5’-end exonuclease digestion (e.g. by exonuclease VIII, RecJf, RecJ, T7 exonuclease, Lambda exonuclease, T5 exonuclease, exonuclease V and / or exonuclease VII).

[0559] The step of digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA may be performed by incubating the combined components under conditions that promote digestion of the precursor dsDNA to produce the digested precursor dsDNA. The digestion of the precursor dsDNA to produce the digested precursor dsDNA may be performed at a first temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or at about 37°C. The digestion may be endonuclease digestion, preferably Type IIS endonuclease digestion.

[0560] The step of ligating the first and second adaptor molecules to the digested precursor dsDNA may be performed by incubating the combined components under conditions that promote ligation of the first and second adaptor molecules to the digested precursor dsDNA to produce the partially protected dsDNA. The ligating may be performed by creating a covalent link between the first and / or second adaptor molecule and the first and / or second end of the digested precursor dsDNA. Preferably, the step of ligation is performed in the presence of T7 DNA ligase.

[0561] The ligation may be at least 5%, at least 10%, at least 15, at least 20%, at least 25%, at least 30%, at least 35%, at least 40, at least 45%, at least 50%, at least 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% efficient. Preferably, the ligation is at least 50%, or at least 70% efficient. For example, at least 5%, at least 10%, at least 15, at least 20%, at least 25%, at least 30%, at least 35%, at least 40, at least 45%, at least 50%, at least 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the digested precursor dsDNA may be incorporated into partially protected dsDNA. Preferably, at least 25 % or at least 30% of the digested precursor dsDNA is incorporated into partially protected dsDNA

[0562] Ligation efficiency may be established based on DNA quantification values before and after the digestion / ligation reaction. Thus, ligation efficiency may be established based on the equation: (starting amplified DNA amount) / (final linear DNA amount) x 100%.

[0563] 51

[0564] 17228500 RGM1 RGM1P262744WQ00

[0565] Ligation efficiency may also be established based on DNA quantification values before and after the digestion / ligation reaction and the subsequent exonuclease treatment to remove remaining open DNA constructs and adaptor molecules excess.

[0566] For example, the precursor dsDNA generated by the amplification (e.g. rolling circle amplification) is first quantified so that the amount of the precursor dsDNA used as the starting material during the digestion / ligation reaction is known. After all the enzymatic reactions, the partially protected dsDNA is quantified to calculate the ligation efficiency as per the equation above.

[0567] DNA quantification methods are known to a person skilled in the art. For example, DNA quantifications may be carried out using the Qubit dsDNA BR assay from ThermoFisher (https: / / www.thermofisher.eom / order / catalog / product / Q32850# / Q32850).

[0568] The step of ligating the digested precursor dsDNA to the first and second adaptor molecules may be performed at a second temperature of 1°C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 11°C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C.

[0569] Using the above conditions the endonuclease may be a Type IIS restriction endonuclease (e.g. Bsal) and the ligase may be T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase. Preferably, the ligase is T7 DNA ligase.

[0570] The step of digesting the second strand of the partially protected dsDNA with an exonuclease (or exonuclease mixture) may be performed at a temperature of 5-90°C, 10-80°C, 15-70°C, 20-60°C, 25-50°C, 30-45°C or 35-40°C. Preferably, the step of digesting the second strand of the partially protected dsDNA with an exonuclease (or exonuclease mixture) is performed at a temperature of 30-45°C. The step of digesting the second strand of the partially protected dsDNA with an exonuclease (or exonuclease mixture) may be performed for at least 10, at least 20, at least 30, at least 40, at least 50, at least 60 or at least 120 minutes. Preferably, the step of digesting the second strand of the partially protected dsDNA with an exonuclease (or exonuclease mixture) may be performed for at least 60 minutes. For example, the step of digesting the second strand of the partially protected dsDNA with an exonuclease (or exonuclease mixture) may be performed at around 37 °C for at least 120 minutes.

[0571] The step of digesting the second strand of the partially protected dsDNA with an exonuclease (or exonuclease mixture) may be followed by inactivating the exonuclease (or exonucleases in the exonuclease mixture), for example by heat-inactivation. Thus, the step of inactivating the exonuclease (or exonucleases in the exonuclease mixture) may be performed at a temperature of 60-90°C, 65-85°C or 70-80°C. Preferably, the step of inactivating the exonuclease (or exonucleases in the exonuclease mixture) is performed at a temperature of 70-80°C. The step of inactivating the exonuclease (or exonucleases in the exonuclease mixture) may be performed at a temperature of at least 60°C, at least 65°C, at least 70°C or at least 80°C. Preferably, the step of inactivating the exonuclease (or

[0572] 52

[0573] 17228500 RGM1 RGM1exonucleases in the exonuclease mixture) is performed at a temperature of at least 70°C. The step of inactivating the exonuclease (or exonucleases in the exonuclease mixture) may be performed for at least 1 , at least 5, at least 10, at least 20 or at least 30 minutes. Preferably, the step of inactivating the exonuclease (or exonucleases in the exonuclease mixture) is performed for at least 5 minutes.

[0574] The step of digesting the second strand of the partially protected dsDNA with an exonuclease (or exonuclease mixture) may be performed at a temperature of 30-45°C for at least 60 minutes followed by performing the step of inactivating the exonuclease (or exonucleases in the exonuclease mixture) at a temperature of at least 70°C for at least 5 minutes.

[0575] The cassette may comprise at least 25, at least 50, at least 100, at least 150, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 325, at least 350, at least 375, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 nucleotides. Preferably, the cassette comprises at least 100 nucleotides.

[0576] The first and second adaptor molecules may be nucleic acids, optionally DNA. The first and second adaptor molecules may comprise a first strand hybridised to a second strand (e.g. dsDNA).

[0577] The first and second adaptor molecules may have different sequences.

[0578] The first and second adaptor molecules may comprise dsDNA comprising a first strand and a second strand. The first strands of the first and second adaptor molecules may be ligated by the ligase to the first strand of the digested precursor dsDNA molecule and the second strands of the first and second adaptor molecules may be ligated by the ligase to the second strand of the digested precursor dsDNA molecule.

[0579] The first adaptor molecule may be compatible with the first end of the digested precursor dsDNA and incompatible with the second end of the digested precursor dsDNA. The second adaptor molecule may be compatible with the second end of the digested precursor dsDNA and incompatible with the first end of the digested precursor dsDNA.

[0580] The first adaptor molecule may be compatible with the 5’ end of the first strand of the digested precursor dsDNA and incompatible with the 3’ end of the first strand of the digested precursor dsDNA. The second adaptor molecule may be compatible with the 3’ end of the first strand of the digested precursor dsDNA and incompatible with the 5’ end of the first strand of the digested precursor dsDNA.

[0581] 53

[0582] 17228500 RGM1 RGM1The first strand of the first adaptor molecule may be compatible with the 5’ end of the first strand of the digested precursor dsDNA and incompatible with the 3’ end of the first strand and the 3’ and 5’ ends of the second strand of the digested precursor dsDNA. The second strand of the first adaptor molecule may be compatible with the 3’ end of the second strand of the digested precursor dsDNA and incompatible with the 5’ end of the second strand and the 3’ and 5’ ends of the first strand of the digested precursor dsDNA.

[0583] The first strand of the second adaptor molecule may be compatible with the 3’ end of the first strand of the digested precursor dsDNA and incompatible with the 5’ end of the first strand and the 3’ and 5’ ends of the second strand of the digested precursor dsDNA. The second strand of the second adaptor molecule may be compatible with the 5’ end of the second strand of the digested precursor dsDNA and incompatible with the 3’ end of the second strand and the 3’ and 5’ ends of the first strand of the digested precursor dsDNA.

[0584] The second strand of the first and second adaptor molecules may not be resistant to exonuclease digestion. The second strand of the first and second adaptor molecules may be susceptible to exonuclease digestion. The second strand of the first and second adaptor molecules may not comprise any nuclease-resistant nucleotides. The second strand may comprise n excisable nucleotides and the second strand may also comprise protected nucleotides. The protected nucleotides may be at the 3’ end of or 3’ of the cassette. The second strand may comprise 1 , 2, 3, 4, 5, 6, 7 or 8 protected nucleotides at the 3’ end or 3’ of the cassette, and there may not be protected nucleotides at the 5’ end of or 5’ of the cassette. The protected nucleotides may be at the 5’ end of or 5’ of the cassette. The second strand may comprise 1 , 2, 3, 4, 5, 6, 7 or 8 protected nucleotides at the 5’ end and there may not be protected nucleotides at the 3’ end or 3’ of the cassette.

[0585] The first strand of the first adaptor molecule may comprise the x nucleotide-resistant nucleotides and the first strand of the second adaptor molecule may comprise the y nucleotide-resistant nucleotides. The nucleotide-resistant nucleotide(s) in the first strand of the first adaptor molecule may be located at the 5’ or 3’ end, preferably the 5’ end; and / or the nucleotide-resistant nucleotide(s) in the first strand of the second adaptor molecule may be located at the 5’ or 3’ end, preferably the 3’ end. The nuclease-resistant nucleotide may be a phosphorothioated nucleotide.

[0586] The second strand of the first adaptor molecule may comprise the x’ nucleotide-resistant nucleotides and the second strand of the second adaptor molecule may comprise the y’ nucleotide-resistant nucleotides. The nucleotide-resistant nucleotide(s) in the second strand of the first adaptor molecule may be located at the 5’ or 3’ end, preferably the 3’ end; and / or the nucleotide-resistant nucleotide(s) in the second strand of the second adaptor molecule may be located at the 5’ or 3’ end, preferably the 5’ end. The nuclease-resistant nucleotide may be a phosphorothioated nucleotide.

[0587] The first adaptor molecule and / or the second adaptor molecule may be a synthetic adaptor molecule.

[0588] 54

[0589] 17228500 RGM1 RGM1The first adaptor molecule and / or the second adaptor molecule may comprise a self-complementary element which creates a loop, such as a hairpin loop or a stem loop. Thus, the first adaptor molecule may comprise a hairpin or a stem-loop. The second adaptor molecule may comprise a hairpin or a stem-loop. Both the first and second adaptor molecules may comprise a hairpin or a stem-loop. The adaptor molecules may each comprise a double-stranded portion comprising a first strand and a second strand, wherein the first strand and the second strand are linked together in a hairpin such that the first strand is hybridized to the second strand. The double-stranded portion of an adaptor may comprise a 3’ overhang or a 5’ overhang of at least 1 , at least 2, at least 3, at least 4, or at least 5 nucleotides. Preferably the 3’ overhang or the 5’ overhang is 4-8 nucleotides. Each end of the digested precursor dsDNA may comprise a 3’ or a 5’ overhang. A portion of the first adaptor molecule (e.g. the overhang) may be complementary to the first end of the digested precursor dsDNA. A portion of the second adaptor molecule may be complementary to the second end of the digested precursor dsDNA.

[0590] If the first and / or second adaptor molecules comprise a hairpin, following ligation of the first and second adaptor molecules to the digested precursor dsDNA there may be a hairpin 3’ and / or 5’ of the cassette. The hairpin may link the first and second strands of the digested precursor dsDNA to form a closed loop at one or both ends. Thus, the methods described herein may further comprise nicking a closed loop at one or both ends in order to generate a partially protected dsDNA with a 3’ and / or 5’ end nucleotide on the second strand.

[0591] The first adaptor molecule and / or the second adaptor molecule may comprise an overhang. The first and / or second ends of the digested precursor dsDNA may comprise a 3’ or a 5’ overhang. A portion of the first adaptor molecule (e.g. the overhang) may be complementary to the first end of the digested precursor dsDNA. A portion of the second adaptor molecule (e.g. the overhang) may be complementary to the second end of the digested precursor dsDNA.

[0592] The first adaptor molecule may comprise an overhang which is complementary to the first end of the digested precursor dsDNA. The second adaptor molecule may comprise an overhang which is complementary to the second end of the digested precursor dsDNA.

[0593] The first adaptor molecule may comprise an overhang which is non-complementary to the second end of the digested precursor dsDNA. The second adaptor molecule may comprise an overhang which is non-complementary to the first end of the digested precursor dsDNA.

[0594] The first adaptor molecule may comprise an overhang complementary to an overhang at the first end of the digested precursor dsDNA and the second adaptor molecule may comprise an overhang complementary to an overhang at the second end of the digested precursor dsDNA. Optionally, the overhang of the first adaptor molecule may not be complementary to the overhang at the second end

[0595] 55

[0596] 17228500 RGM1 RGM1of the digested precursor dsDNA and the overhang of the second adaptor molecule may not be complementary to the overhang at the first end of the digested precursor dsDNA.

[0597] The first adaptor molecule may comprise an overhang that is complementary to and anneals to a 5’ overhang of the digested precursor dsDNA and the second adaptor molecule may comprise an overhang that is complementary to and anneals to a 3’ overhang of the digested precursor dsDNA.

[0598] The first adaptor molecule may comprise an overhang that is complementary to and anneals to a 3’ overhang of the digested precursor dsDNA and the second adaptor molecule may comprise an overhang that is complementary to and anneals to a 5’ overhang of the digested precursor dsDNA.

[0599] The first adaptor molecule and / or the second adaptor molecule may not be a plasmid or a vector DNA.

[0600] The first adaptor molecule and / or the second adaptor molecule may comprise a single-stranded portion. The single-stranded portion may form a hairpin or a stem-loop. Thus, the first adaptor molecule and / or the second adaptor molecule may comprise a loop portion. The single-stranded portion may comprise less than 10, 9, 8, 7, 6, 5, 4, 3, 2 nucleotides. Preferably, the single-stranded portion comprises 5 nucleotides.

[0601] The first adaptor molecule and / or the second adaptor molecule may comprise a double-stranded portion. The double-stranded portion may comprise less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, or less than 10 base pairs. The doublestranded portion may comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, or at least 15 base pairs.

[0602] The first adaptor molecule and / or the second adaptor molecule may comprise a 5’ phosphate. The 5’ phosphate may facilitate ligation to the digested precursor dsDNA (which may comprise a 3’-OH group at first and / or second ends). The first adaptor molecule and / or the second adaptor molecule may comprise a 3’-OH. The 3’-OH may facilitate ligation to the digested precursor dsDNA (which may comprise a 5’ phosphate at first and / or second ends).

[0603] The first adaptor molecule may comprise a portion that is complementary to the first end of the digested precursor dsDNA. The second adaptor molecule may comprise a portion that is complementary to the second end of the digested precursor dsDNA. The first adaptor molecule may comprise a portion that anneals to the first end of the digested precursor dsDNA. The second adaptor molecule may comprise a portion that anneals to the second end of digested precursor dsDNA. The first adaptor molecule may comprise a portion that is complementary and anneals to the first end of the digested precursor dsDNA. The second adaptor molecule may comprise a portion that is complementary and anneals to the second end of the digested precursor dsDNA.

[0604] 56

[0605] 17228500 RGM1 RGM1The portion that is complementary or anneals to the first or second end of the digested precursor dsDNA may be a 5’ overhang or a 3’ overhang of the first and / or second adaptor molecule. The overhang of the first adaptor molecule may be complementary to the first end of the digested precursor dsDNA and / or the overhang of the second adaptor molecule may be complementary to the second end of the digested precursor dsDNA. The overhang of the first adaptor molecule may anneal to the first end of the digested precursor dsDNA and / or the overhang of the second adaptor molecule may anneal to the second end of the digested precursor dsDNA. The overhang of the first adaptor molecule may be complementary to and anneal to the first end of the digested precursor dsDNA and / or the overhang of the second adaptor molecule may be complementary to and anneal to the second end of the digested precursor dsDNA.

[0606] The first adaptor molecule may be appended to a first end of the digested precursor dsDNA and the second adaptor molecule may be appended to a second end of the digested precursor dsDNA. The appending of the first adaptor molecule and / or the second adaptor molecule may be performed by hybridization or ligation of the adaptor molecules to the ends of the digested precursor dsDNA. Thus, the first adaptor molecule may be hybridized to the first end of the digested precursor dsDNA. The second adaptor molecule may be hybridized to the second end of the digested precursor dsDNA. The first adaptor molecule may be ligated to the first end of the digested precursor dsDNA. The second adaptor molecule may be ligated to the second end of the digested precursor dsDNA. The appending of the first adaptor molecule and the second adaptor molecule may be performed by both hybridization and ligation of the adaptor molecules to the ends of the digested precursor dsDNA. Thus, the first adaptor molecule may be hybridized and ligated to the first end of the digested precursor dsDNA. The second adaptor molecule may be hybridized and ligated to the second end of the digested precursor dsDNA. The hybridization is based on complementarity of a portion of the first and / or second adaptor molecules to the first and / or second end of the digested precursor dsDNA.

[0607] The first adaptor molecule and / or the second adaptor molecule may not comprise a Type IIS endonuclease target sequence. The first adaptor molecule and / or the second adaptor molecule may not comprise Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsell, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV11, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104l, Earl, Ecil, Eco31l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp11091, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequences.

[0608] The first adaptor molecule and / or the second adaptor molecule may comprise one or more locked nucleic acids (LNAs).

[0609] 57

[0610] 17228500 RGM1 RGM1The first adaptor molecule and / or the second adaptor molecule may confer resistance to the nuclease digestion, such as exonuclease digestion (e.g. exonuclease III or VIII digestion).

[0611] The first adaptor molecule and / or the second adaptor molecule may comprise one or more protected nucleotides (i.e. nuclease-resistant nucleotides), such as phosphorothioated nucleotides. The protected nucleotides may be located in the single-stranded portion (e.g. hairpin portion) or the double-stranded portion. The protected nucleotides may be located in the overhang portion of the adaptor molecules.

[0612] The first and second adaptor molecules may comprise one or more phosphorothioated nucleotides, such that, once the adaptor molecules are appended (e.g. ligated) to the digested precursor dsDNA and the second strand of the partially protected dsDNA is digested by the exonuclease (or exonuclease mixture), the protected DNA is resistant to nuclease digestion or has improved or enhanced resistance to nuclease digestion. The protected DNA product may be resistant to 3’-end exonuclease digestion (e.g. by exonuclease III) and / or 5’-end exonuclease digestion (e.g. by exonuclease VIII).

[0613] The adaptor molecule may comprise a plurality of phosphorothioated nucleotides. For example, the adaptor molecules may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15 or at least 16 phosphorothioated nucleotides in each strand. The first and / or second adaptor molecule may comprise protected nucleotides in the first strand and may not comprise protected nucleotides in the second strand.

[0614] The adaptor molecule may be a nucleic acid adaptor molecule. The adaptor molecule may be doublestranded. The adaptor molecule may comprise a portion that is double-stranded.

[0615] The first and / or second adaptor molecules may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, or at least 16 base pairs.

[0616] The adaptor molecule may comprise a plurality of phosphorothioated nucleotides at internal positions in each strand. For example, the adaptor molecules may comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15 or at least 16 phosphorothioated nucleotides at internal positions in each strand. Preferably, the adaptor molecule comprises at least 2 phosphorothioated nucleotides at internal positions in each strand.

[0617] The internal positions may not be located between the second and penultimate nucleotide of the adaptor molecule. The internal positions may be any position in the adaptor molecules other than the last nucleotide at the end of each strand.

[0618] 58

[0619] 17228500 RGM1 RGM1The adaptor molecule may comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of protected nucleotides.

[0620] Once the adaptor molecules are appended to the digested precursor dsDNA and the partially protected dsDNA is digested by the exonuclease (or exonuclease mixture), the protected DNA may comprise a protected nucleotide at the 5’ end of the ssDNA cassette or 5’ of the ssDNA cassette, and a nuclease-resistant nucleotide at the 3’-end of the ssDNA cassette or 3’ of the ssDNA cassette.

[0621] The partially protected dsDNA may be generated from the precursor dsDNA by performing the steps described herein in a single reaction (i.e. as a single step) in a single contiguous aqueous volume. For example, the steps of contacting a precursor dsDNA with an endonuclease, digesting the precursor dsDNA with the endonuclease to generate a digested precursor dsDNA, contacting the digested precursor dsDNA with a ligase and first and second adaptor molecules, and ligating the first and second adaptor molecules to the digested precursor DNA thereby generating the partially protected dsDNA, may be performed in a single reaction (i.e. as a single step) in a single contiguous aqueous volume. Thus, the partially protected dsDNA may be generated by incubating the precursor dsDNA with the endonuclease, the ligase and first and second adaptor molecules in a single reaction (i.e. a single step) in a single contiguous aqueous volume. The single contiguous aqueous volume may be incubated in order to allow the required digestion and ligation.

[0622] The single contiguous aqueous volume may be incubated to generate the partially protected dsDNA by digesting the precursor dsDNA and ligating the first and second adaptor molecules to the digested precursor dsDNA.

[0623] The step of incubating the single contiguous aqueous volume may be performed under conditions that promote ligation of the first and second adaptor molecules to the digested precursor dsDNA to produce the partially protected dsDNA. The appending may be performed by creating a covalent link between the first and / or second adaptor molecule and the end(s) of the digested precursor dsDNA.

[0624] The step of incubating the single contiguous aqueous volume may be performed under conditions that promote digestion of the precursor dsDNA to produce the digested precursor dsDNA. The digestion of the precursor dsDNA to produce the digested precursor dsDNA may be performed at a first temperature of 1°C-100°C, 1°C -80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C -45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or at about 37°C. The digestion may be endonuclease digestion, preferably Type IIS endonuclease digestion.

[0625] The step of incubating the single contiguous aqueous volume may be performed under conditions that promote ligation of the digested precursor dsDNA to the first and second adaptor molecules. The ligation may be at least 5%, at least 10%, at least 15, at least 20%, at least 25%, at least 30%, at least

[0626] 59

[0627] 17228500 RGM1 RGM135%, at least 40, at least 45%, at least 50%, at least 55%, at least 60, at least 65, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% efficient. For example, at least 5%, at least 10%, at least 15, at least 20%, at least 25%, at least 30%, at least 35%, at least 40, at least 45%, at least 50%, at least 55%, at least 60, at least 65, at least 70%, at least 75, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the digested precursor dsDNA may be incorporated into partially protected dsDNA. Preferably, the ligation is at least 15% efficient.

[0628] The step of ligation of the digested precursor dsDNA to the first and second adaptor molecules may be performed at a second temperature of 1°C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 11°C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C.

[0629] The step of incubating the single contiguous aqueous volume may comprise incubating at a first temperature and then incubating at a second temperature. The first temperature may be 1°C-100°C, 1°C-80°C, 5°C-70°C, 10°C-60°C, 15°C-55°C, 20°C-50°C, 25°C-45°C, 30°C-40°C, 35°C-39°C, 36°C-38°C, or about 37°C. The second temperature may be 1°C -90°C, 2°C -70°C, 5°C-60°C, 8°C-55°C, 9°C-50°C, 10°C-45°C, 11°C-40°C, 12°C-37°C, 13°C-30°C, 14°C-25°C, 15°C-20°C or at about 16°C. Preferably, the first temperature is 35°C-39°C. Preferably, the second temperature is 14°C-18°C.

[0630] The step of incubating the single contiguous aqueous volume may be performed isothermally. The step of incubating the single contiguous aqueous volume may comprise incubating at a constant temperature. The constant temperature promotes simultaneous digestion of the double-stranded DNA molecule to produce the linear portion of the double-stranded DNA molecule and ligation of the linear double-stranded region to the first and second adaptor molecules. For example, the constant temperature may be 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. Preferably, the constant temperature is 30°C. The constant temperature is intended to mean that the temperature does not significantly change during the reaction. The constant temperature is intended to mean that the temperature variation during the step of incubating the single contiguous aqueous volume is less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1°C. In a preferred embodiment the temperature during the step of incubating the single contiguous aqueous does not deviate by more than 5°C, preferably by not more than 3°C, even more preferably not more than 1 °C. Thus, the constant temperature may be a temperature in a range of 20°C-30°C, 22°C-32°C, 24°C-34°C, 26°C-36°C, 28°C-38°C, 30°C-40°C, 22°C-28°C, 32°C-38°C, 25°C-35°C, 26°C-34°C, 27°C- 33°C, 27.5°C-32.5°C, 28°C-32°C, 28.5°C-31 ,5°C, 29°C-31°C, or 29.5°C-30.5°C. Preferably, the constant temperature is a temperature in a range of 27.5°C-32.5°C. Alternatively, the constant temperature may be a temperature in a range of 32°C-42°C, 33°C-41°C, 34°C-40°C, 35°C-39°C, 36°C-38°C. Preferably, the constant temperature is a temperature in a range of 34.5°C-39.5°C.

[0631] The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature. The step of incubating the single contiguous aqueous volume

[0632] 60

[0633] 17228500 RGM1 RGM1may comprise cycling between the first temperature and the second temperature at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 80, at least 90, or at least 100 times, preferably at least 20 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature less than 40, less than 35, less than 30 times, less than 29, less than 25 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature 2-100, 5-80, 10-70, 20-60, or 30-60 times. The step of incubating the single contiguous aqueous volume may comprise cycling between the first temperature and the second temperature 2-20, 5-29, 61-100, or 65-80 times.

[0634] The DNA template may comprise at least one endonuclease target sequence. Thus, the DNA template may comprise at least one endonuclease target sequence. Preferably, the DNA template comprises at least two endonuclease target sequences. The endonuclease target sequences may be the same or different. Preferably, the at least one endonuclease target sequence is a restriction endonuclease target sequence. Different restriction endonuclease target sequences would be known to the skilled person. The endonuclease target sequence may be a Type IIS restriction endonuclease target sequence. For example, the restriction endonuclease target sequence may be a Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3l,Sapl, Aarl, Acc36l, AcIWI, Acul, Ajul, Alol, Alw26l, Alwl, Arsl, AsuHPI, Bael, Bari, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAI, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bsell, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, Bsgl, BsIFI, BsmAI, BsmFI, Bsml, Bso31 l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6l, BstF5l, BstMAI, BstV11, BstV2l, Bsul, BtgZI, BtsCI, Btsl-v2, BtsMutl, Bvel, Csel, CspCI, Eam1104l, Earl, Ecil, Eco31l, Eco57l, Esp3l, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp11091, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI target sequence. The at least one endonuclease target sequence may be a native endonuclease sequence (i.e. an endonuclease sequence present in the template molecule). Alternatively, the at least one endonuclease target sequence may be introduced to the DNA template molecule prior to the production of the protected DNA.

[0635] The DNA template used in the methods described herein may be single-stranded or double-stranded. Preferably, the DNA template is double-stranded. The DNA template may be a natural circular DNA molecule. For example, the DNA template molecule may be (i) a plasmid, (ii) a minicircle, (iii) a cosmid, (iv) a bacterial artificial chromosome (BAC), or (v) a molecular inversion probe (MIP). The DNA template molecule may be an enzymatically produced circular DNA molecule. For example, the DNA template may be (i) a circular DNA molecule obtained from recombinase reaction, preferably Cre recombinase reaction, or (ii) a circular DNA molecule obtained from ligase reaction, preferably using the golden gate assembly. The DNA template may be an enzymatically produced covalently-closed linear DNA molecule. For example, the DNA template may be (i) a DNA molecule processed with TelN protelomerase; or (ii) a DNA molecule generated by ligation of the DNA ends with an adaptor. The DNA

[0636] 61

[0637] 17228500 RGM1 RGM1template molecule may comprise an element that is double-stranded and an element that is singlestranded. For example, the DNA template may comprise a double-stranded DNA and a single-stranded hairpin loop.

[0638] The DNA template may be linear. If the DNA template is linear, prior to amplification (e.g. rolling circle amplification), a DNA template may be circularized to produce a DNA template suitable for use in the methods described herein.

[0639] The digested precursor dsDNA may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11 ,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the digested precursor dsDNA is at least 200 base pairs long.

[0640] The first end of the digested precursor dsDNA may be complementary to a portion of the first adaptor molecule. The second end of the digested precursor dsDNA may be complementary to a portion of the second adaptor molecule. The first end and / or the second end of the digested precursor dsDNA may be generated by endonuclease digestion.

[0641] The digested precursor dsDNA may comprise a 3’-OH group at first and / or second ends. The 3’-OH group may facilitate ligation to the first and / or second adaptor molecule(s) (which may comprise a 5’ phosphate). The digested precursor dsDNA may comprise a 5’ phosphate at first and / or second ends. The 5’ phosphate may facilitate ligation to the first and / or second adaptor molecule(s) (which may comprise a 3’-OH group).

[0642] The digested precursor dsDNA may comprise an overhang. For example, the digested precursor dsDNA may comprise a 5’ overhang or a 3’ overhang. The digested precursor dsDNA may comprise a blunt end or blunt ends. The digested precursor dsDNA may comprise: a 5’ overhang and a blunt end, two 5’ overhangs, a 3’ overhang and a blunt end, two 3’ overhangs, or a 5’ overhang and a 3’ overhang. The overhang may have at least 3 nucleotides (preferably from 4 to 8 nucleotides). The overhang may be in the first or second strand of the digested precursor dsDNA.

[0643] 4. Methods for producing an RNA product using an adaptor-ligated DNA product and a protected splint DNA

[0644] The methods provides herein may be combined to allow the more efficient production of an RNA product.

[0645] The invention provides a method for producing an RNA product, wherein the method comprises: (I) amplifying a DNA template molecule, wherein the DNA template molecule comprises at least one endonuclease target sequence to generate an amplified DNA product, optionally wherein the DNA template molecule is amplified by any of the methods described herein;

[0646] 62

[0647] 17228500 RGM1 RGM1(II) producing an adaptor-ligated DNA product as a template for in vitro transcription (IVT), wherein the adaptor-ligated DNA product is produced from the amplified DNA product, optionally wherein the adaptor-ligated DNA product is produced from the amplified DNA product using any of the methods described herein;

[0648] (III) producing a protected splint DNA, wherein the protected splint DNA is produced from the amplified DNA product, optionally wherein the protected splint DNA is produced from the amplified DNA product using any of the methods described herein (i.e. the amplified DNA product is a precursor dsDNA); (IV) providing a first RNA molecule, a second RNA molecule and a third RNA molecule, wherein the first RNA molecule is a chemically synthesized RNA molecule, the second RNA molecule is produced by in vitro transcription (IVT) using the adaptor-ligated DNA product as template for the in vitro transcription (IVT), and the third RNA molecule is a chemically synthesized RNA molecule; and (V) annealing the first, second and third RNA molecules to the protected splint DNA; and ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule.

[0649] The invention provides a method for producing an RNA product, wherein the method comprises: (I) amplifying a DNA template molecule, wherein the DNA template molecule comprises at least one endonuclease target sequence to generate amplified DNA product, optionally wherein the DNA template molecule is amplified by any of the methods described herein;

[0650] (II) producing an adaptor-ligated DNA product as a template for in vitro transcription (IVT), wherein the adaptor-ligated DNA product is produced from the amplified DNA product, optionally wherein the adaptor-ligated DNA product is produced from the amplified DNA product using any of the methods described herein;

[0651] (III) producing a protected splint DNA, wherein the protected splint DNA is produced from the amplified DNA product, optionally wherein the protected splint DNA is produced from the amplified DNA product using any of the methods described herein (i.e. the amplified DNA product is a precursor dsDNA); (IV) producing a second RNA by in vitro transcription (IVT) using the adaptor-ligated DNA product as a template for in vitro transcription (IVT);

[0652] (V) providing a first RNA molecule and a third RNA molecule, wherein the first RNA molecule is a chemically synthesized RNA molecule and the third RNA molecule is a chemically synthesized RNA molecule; and

[0653] (VI) annealing the first, second and third RNA molecules to the protected splint DNA; and ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule.

[0654] The step of amplifying a DNA template may be an in vitro or in vivo amplification. Preferably, the amplification is an in vitro amplification. For example, the amplification may be performed by rolling circle amplification (RCA), MALBAC method, traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-

[0655] 63

[0656] 17228500 RGM1 RGM1dependent amplification (HDA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). Preferably, the amplification is rolling circle amplification (RCA).

[0657] Rolling circle amplification may be performed without any primers, or in the presence of a primer or multiple primers. For example, the primer may be a synthetic primer. The primers may be random primers. Rolling circle amplification may be performed in the presence of a primase. The primase may be TthPrimPol. Preferably, if the rolling circle amplification is performed without any primers, it is performed in the presence of a primase, such as TthPrimPol. Similarly, if a primer is used during amplification reaction, a primase is not used. The double-stranded DNA product may be generated by the rolling circle amplification in vitro under isothermal conditions using a suitable nucleic acid polymerase, such as Phi29 DNA polymerase.

[0658] The step of producing an adaptor-ligated DNA product as a template for in vitro transcription (IVT) may be performed using first and second adaptor molecules. The first and second adaptor molecules may be any of the first and second adaptor molecules described herein in relation to the production of an adaptor-ligated DNA product.

[0659] The step of producing a protected splint DNA may be performed using first and second adaptor molecules. The first and second adaptor molecules may be any of the first and second adaptor molecules described herein in relation to the production of a protected splint DNA.

[0660] The first and second adaptor molecules used to produce an adaptor-ligated DNA product may be different to the first and second adaptor molecules used to produce a protected splint DNA. Thus, the first and second adaptor molecules used to produce a protected splint DNA may also be referred to herein as third and fourth adaptor molecules.

[0661] The method may further comprise digesting the splint DNA using DNase. The DNase may be DNase I and / or Duplex DNase.

[0662] The method may further comprise digesting the unligated RNA molecules using one or more RNA exonucleases. The RNA exonuclease(s) may be a 5’-3’ RNA exonuclease and / or a 3’-5’ RNA exonuclease. The one or more RNA exonucleases may be selected from XRN-I (5’ to 3’), Terminator 5'-Phosphate-Dependent Exonuclease (5’ to 3’), Exonuclease T (3’ to 5’), Exolll (3’ to 5’) and / or RNase R (3’ to 5’).

[0663] 5. Use of nuclease-resistant nucleotides (or protected nucleotides) in the methods and products

[0664] The RNA molecules and / or the DNA molecules (e.g. the DNA splint) may comprise one or more nuclease-resistant (or protected nucleotides). Preferably, the RNA molecules and / or the DNA molecules (e.g. the DNA splint) may each comprise two or more nuclease-resistant (or protected

[0665] 64

[0666] 17228500 RGM1 RGM1nucleotides). As used herein the term “nuclease-resistant nucleotide” or “protected nucleotide” is intended to encompass any type of nucleotide that provides or enhances resistance to nuclease digestion (especially exonuclease digestion).

[0667] The nuclease-resistant nucleotide(s) (i.e. a nucleotide(s) resistant to exonuclease digestion) may be a phosphorothioated nucleotide (PS). As used herein, the term "phosphorothioated nucleotide" refers to a nucleotide that has an altered phosphate backbone, wherein the sugar moieties are linked by a phosphorothioate bond. In the phosphate backbone of an oligonucleotide sequence, the phosphorothioate bond contains a sulphur atom as a substitute for a non-bridging oxygen atom. This modification renders the internucleotide linkage resistant to nuclease degradation.

[0668] The phosphorothioated nucleotide(s) may comprise a-S-dATP (i.e. 2’-deoxyadenosine-5’-(a-thio)-triphosphate), a-S-dCTP (i.e. 2’-deoxycytidine-5’-(a-thio)-triphosphate), a-S-dGTP (i.e. 2’-deoxyguanosine-5’-(a-thio)-triphosphate), a-S-dTTP (i.e. 2’-deoxythymidine-5’-(a-thio)-triphosphate), a-S-dUTP (i.e. 2’-deoxyuridine-5’-(a-thio)-triphosphate), and / or uridine 2’, 3’-cyclophosphorothioate.

[0669] The phosphorothioated nucleotide(s) may be Sp-isomers, Rp-isomers or a mixture of both Sp- and Rp-isomers.

[0670] The nuclease-resistant nucleotide(s) (i.e. nucleotide(s) resistant to exonuclease digestion) may be 2'-O-methyl (2’ O-Me) nucleotides or 2'-O-methoxyethyl (MOE) nucleotides.

[0671] The nuclease-resistant nucleotide(s) may be 2’ O-Me nucleotides of at least one type, or at least two, at least three or at least four types. For example, the 2’ O-Me nucleotide(s) may comprise one or more of the following nucleosides: 2’-O-methyl adenosine, 2’-O-methyl cytidine, 2’-O-methyl guanosine, 2’-O-methyl thymidine and / or 2’-O-methyl uridine.

[0672] The nuclease-resistant nucleotide(s) may be MOE nucleotide(s) of at least one type, or at least two, at least three or at least four types. For example, the MOE nucleotide(s) may comprise one or more of the following nucleosides: 2’-O-methoxy-ethyl adenosine, 2’-O-methoxy-ethyl cytidine, 2’-O-methoxy-ethyl guanosine, 2’-O-methoxy-ethyl thymidine and / or 2’-O-methoxy-ethyl uridine.

[0673] The nuclease-resistant nucleotide(s) may comprise 5-Methylcytidine(s).

[0674] The nuclease-resistant nucleotide(s) may be locked nucleic acid(s) (LNA).

[0675] The nuclease-resistant nucleotide(s) may comprise pseudouridine(s) and / or N1-Methyl-Pseudouridine(s).

[0676] The nuclease-resistant nucleotide(s) may comprise phosphorothioated nucleotide(s) and 2’-O-methyl nucleotide(s). Any single nuclease-resistant nucleotide may comprise both phosphorothioate and 2’-O-

[0677] 65

[0678] 17228500 RGM1 RGM1methyl modifications. The first and third RNA molecules may both comprise phosphorothioate modifications. The first and third RNA molecules may both comprise 2’-O-methyl modifications. The first and third RNA molecules may both comprise phosphorothioate modifications and 2’-O-methyl modifications. The first and third RNA molecules may both comprise single nuclease-resistant nucleotides that comprise both phosphorothioate modifications and 2’-O-methyl modifications.

[0679] 6. RNA products

[0680] The invention provides an RNA product as described herein.

[0681] The RNA product may be a linear RNA product.

[0682] The RNA product may comprise a long RNA, a long non-coding RNA (IncRNA), a non-coding RNA (ncRNA), a guide RNA (gRNA), a single guide RNA (sgRNA), a prime editing guide RNA (pegRNA), base-editing guide RNA or an mRNA (e.g. an mRNA encoding a pathogen-specific antigen or an mRNA encoding a neoantigen). The gRNA may comprise an aptamer sequence (e.g. MS2 aptamer sequence).

[0683] The RNA product may comprise an RNA cassette. The RNA cassette may comprise a long RNA, a long non-coding RNA (IncRNA), a non-coding RNA (ncRNA), a guide RNA (gRNA), a prime editing guide RNA (pegRNA) or an mRNA (e.g. an mRNA encoding a pathogen-specific antigen or an mRNA encoding a neoantigen). The gRNA may comprise an aptamer sequence (e.g. MS2 aptamer sequence).

[0684] The RNA product may be at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, at least 105 nucleotides, at least 110 nucleotides, at least 115 nucleotides, at least 120 nucleotides, at least 125 nucleotides, at least 135 nucleotides, at least 145 nucleotides, at least 150 nucleotides, at least 155 nucleotides, at least 160 nucleotides, at least 165 nucleotides, at least 170 nucleotides, at least 175 nucleotides, at least 180 nucleotides, at least 185 nucleotides, at least 190 nucleotides, at least 195 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 750 nucleotides, at least 1000 nucleotides, at least 1500 nucleotides, at least 2000 nucleotides, at least 3000 nucleotides, at least 4000 nucleotides, at least 5000 nucleotides, at least 6000 nucleotides, at least 7000 nucleotides, at least 8000 nucleotides or at least 9000 nucleotides. Preferably, the RNA product is at least 135 nucleotides.

[0685] The RNA product may be a protected linear RNA. The RNA product may comprise two or more nuclease resistant nucleotides (i.e. nucleotides resistant to exonuclease digestion). The RNA product may be resistant to 5’-3’ exonuclease digestion and / or 3’-5’ exonuclease digestion. The RNA product may comprise at least 1 nuclease resistant nucleotide 5’ of the RNA cassette and at least 1 nuclease resistant nucleotide 3’ of the RNA cassette. The RNA product may comprise at least 2 nuclease resistant nucleotides 5’ of the RNA cassette and at least 2 nuclease resistant nucleotides 3’ of the RNA

[0686] 66

[0687] 17228500 RGM1 RGM1cassette. The RNA product may comprise at least 3 nuclease resistant nucleotides 5’ of the RNA cassette and at least 3 nuclease resistant nucleotides 3’ of the RNA cassette. The RNA product may comprise at least 4 nuclease resistant nucleotides 5’ of the RNA cassette and at least 4 nuclease resistant nucleotides 3’ of the RNA cassette. The RNA product may comprise at least 5 nuclease resistant nucleotides 5’ of the RNA cassette and at least 5 nuclease resistant nucleotides 3’ of the RNA cassette. Preferably, the RNA product comprises at least 2 nuclease resistant nucleotides 5’ of the RNA cassette and at least 2 nuclease resistant nucleotides 3’ of the RNA cassette.

[0688] The invention provides an RNA product (e.g. a guide RNA or a pegRNA) produced or obtainable by the methods for producing an RNA product as described herein.

[0689] BRIEF DESCRIPTION OF THE DRAWINGS

[0690] FIG. 1 (A) Schematic illustrating the components of a prime editing (PE) ribonucleoprotein complex (RNP) and a target DNA. The PE RNP includes a prime editing guide RNA (pegRNA and a fusion protein of Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV) and Streptococcus pyogenes Cas9 nickase (nSpCas9). The positions of the Primer Binding Site (PBS) template, Reverse Transcription (RT) template and Spacer on the pegRNA are shown. (B) Schematic of a pegRNA illustrating from 5’ to 3’ the following sequence elements (and their sizes): Spacer, CRISPR RNA (crRNA)Ztrans-activating CRISPR RNA (tracRNA), Reverse Transcription (RT) template and Primer Binding Site (PBS) template.

[0691] FIG. 2 Schematic illustrating a method for producing a pegRNA. In the method, rolling circle amplification (RCA) is used to produce a concatemer comprising sequences of a pegRNA. The concatemer is then added to a digestion and ligation reaction (DL) with first and second adaptor molecules; in each case, the first adaptor molecule is ligated to a first end of a digested monomer of the concatemer and the second adaptor molecule is ligated to a second of a digested monomer of the concatemer. A first set of adaptor molecules are used in the DL reaction on the left side of the figure to produce a double-stranded DNA molecule with a lower strand with protected nucleotides at either end. Exonucleases are then used to digest the upper strand to produce a single-stranded DNA (ssDNA) that will serve as the splint DNA. A second set of adaptor molecules are used in the DL reaction on the right side of the figure to produce a double-stranded DNA molecule with a T7 promoter (T7 pro). This double-stranded DNA molecule is then used as a template for in vitro transcription (IVT) to produce an RNA comprising crRNA / tracRNA and RT template sequences (this is an example of a second RNA molecule). In the final step, a first RNA molecule (RNA Oligo Spacer PS and / or 2 O-Me) and a third RNA molecule (RNA Oligo RTT / PBS PS and / or 2 O-Me) are ligated to either end of the second RNA molecule by splint ligation using the splint DNA. In this example, the first RNA molecule and the third RNA molecule have been chemically synthesized.

[0692] 67

[0693] 17228500 RGM1 RGM1P262744WQ00

[0694] FIG. 3 Schematic illustrating a method for producing a pegRNA. In the method, a double-stranded DNA molecule with a T7 promoter (T7 pro) is used as a template for in vitro transcription (IVT) to produce an RNA comprising crRNA / tracRNA and RT template sequences (this is an example of a second RNA molecule). In addition, a first RNA molecule (RNA Oligo Spacer PS and / or 2 O-Me), a third RNA molecule (RNA Oligo RTT / PBS PS and / or 2 O-Me) and a splint DNA are chemically synthesized. In the final step, the first RNA molecule and the third RNA molecule are ligated to either end of the second RNA molecule by splint ligation using the splint DNA.

[0695] FIG. 4 Schematic illustrating a splint ligation reaction (formed of an RNA / RNA hybrid) followed by DNase treatment to remove the DNA strand leaving a single-stranded pegRNA.

[0696] FIG. 5 Schematic illustrating how all unligated products are degraded by exonuclease because they are not protected on both ends. Only the fully ligated product formed of three RNA molecules (as illustrated in FIG. 2 and 3) has protected nucleotides at both ends and is resistant to degradation by exonucleases.

[0697] FIG. 6 Schematic of the three RNA molecule splint ligation strategy for generating pegRNAs. The first RNA molecule (5’RNA Oligo 20nt; SEQ ID NO: 1) is a chemically synthesized 20 nucleotide phosphorothioated (PS) RNA encoding a spacer sequence. The second RNA molecule (IVT RNA 108nt; SEQ ID NO: 2) is 108 nucleotide RNA synthesized by in vitro transcription (IVT) encoding a scaffold region and a first portion of a Reverse transcription template (RTT). The third RNA molecule (3’RNA Oligo 16nt; SEQ ID NO: 3) is a chemically synthesized 16 nucleotide phosphorothioated (PS) RNA encoding a second portion of the RTT and a primer binding site (PBS). The figure shows the three RNA molecules annealed to a splint DNA. Ligation is performed using T4 RNA ligase 2 followed by DNase I treatment to degrade the splint DNA leaving the 144 nucleotide single-stranded pegRNA product (Ligated pegRNA 144nt).

[0698] FIG. 72% denaturing agarose gel of three molecule ligation products: IVT pegRNA 108nt indicates an unligated second RNA molecule (i.e. a 108 nucleotide synthesized by in vitro transcription (IVT) encoding a scaffold region and a first portion of a Reverse transcription template (RTT)); 5’RNA oligo spacer 20nt indicates an unligated first RNA molecule (i.e. a chemically synthesized 20 nucleotide phosphorothioated (PS) RNA encoding a spacer sequence); 3’RNA oligo RTT 16nt indicates an unligated third RNA molecule (i.e. a chemically synthesized 16 nucleotide phosphorothioated (PS) RNA encoding a second portion of the RTT and a primer binding site (PBS)); IVT pegRNA 146nt indicates the full-length IVT synthesized size-control RNA; and Ligated pegRNA 144nt indicates full-length pegRNA (144 nucleotides) formed of first, second and third RNA molecules (as illustrated in FIG.6). On the left side of the figure is an annotated version of the ssRNA ladder shown in the main image.

[0699] FIG. 8 15% denaturing Urea PAGE of three RNA molecule ligation products: 5’RNA oligo spacer 20nt indicates an unligated first RNA molecule (i.e. a chemically synthesized 20 nucleotide

[0700] 68

[0701] 17228500 RGM1 RGM1phosphorothioated (PS) RNA encoding a spacer sequence); IVT RNA 108 nt indicates an unligated second RNA molecule (i.e. a 108 nucleotide synthesized by in vitro transcription (IVT) encoding a scaffold region and a first portion of a Reverse transcription template (RTT)); IVT pegRNA 146nt indicates the full-length IVT synthesized size-control RNA; Ligated pegRNA 144 nt indicates full-length pegRNA (144 nucleotides) formed of first, second and third RNA molecules (as illustrated in FIG.6); Low ssRNA ladder; and 3’RNA oligo RTT 16nt indicates an unligated third RNA molecule (i.e. a chemically synthesized 16 nucleotide phosphorothioated (PS) RNA encoding a second portion of the RTT and a primer binding site (PBS)). On the right side of the figure is an annotated version of the Low ssRNA ladder shown in the main image.

[0702] FIG. 9 Overview of sgRNA / pegRNA ligation strategy and synthesis. A) three-fragment ligation strategy using a chemically synthesized first RNA molecule (5’ Oligo; 19 nt) and a chemically synthesized third RNA molecule (3’ Oligo; 20 nt) which contain phoshorothioated (PS) and 2’-O-methylated (2’ O-Me) modified nucleotides. The second RNA molecule (IVT RNA) is a 89 nt RNA molecule synthesized by in vitro transcription (IVT). The first, second and third RNA molecules may be ligated together on a splint DNA, using the approach illustrated on the left-hand side in Figure 10. B) 10% denaturing Urea PAGE of sgRNA / pegRNA products synthesized using different strategies. Lane 2, indicates full-length ligated sgRNA / pegRNA (128 nt) comprising a second RNA molecule (IVT RNA; 89 nt) which has been synthesized by IVT ligated to a chemically synthesized first RNA molecule (5’ Oligo; 19 nt) and a chemically synthesized third RNA molecule (3’ Oligo; 20 nt). Lane 3, indicates full-length ligated sgRNA / pegRNA (128 nt) comprising a second RNA molecule (SPOS RNA; 89 nt) which has been chemically synthesized and has been ligated to the same first and third RNA molecules as in Lane 2. Lane 4, indicates full length sgRNA / pegRNA (128 nt) produced entirely via chemical synthesis with identical modifications to the RNA molecule of Lane 2. C) Schematic of the strategies for synthesis of the sgRNA / pegRNA molecules (128 nt) which are analysed in Lanes 2, 3 and 4 of B). D) Densitometric analysis of the sgRNA / pegRNA products in Lanes 2, 3 and 4 displayed as % sgRNA (128 nt) of full product per lane.

[0703] FIG. 10 Schematic of the strategies for synthesizing sgRNA / pegRNA via ligation of a second RNA molecule synthesized via IVT (IVT RNA) to chemically synthesized first and third RNA molecules (5’ Oligo and 3’ Oligo). Outlined are different strategies which rely on enzymatic processing of the second RNA molecule (IVT RNA) and ligation of the first and third RNA molecules (5’ Oligo and 3’ Oligo) to the second RNA molecule (IVT RNA) by: (1) ssDNA splint-based ligation using T4 RNA ligase 2 (reaction scheme on left hand side); (2) standard ligation using T4 RNA ligase 1 (indicated by curved arrow on right hand side); or (3) a combination of standard ligation of the third RNA molecule (3’ Oligo) to the second RNA molecule (IVT RNA) using T4 RNA ligase 1 followed by ssDNA splint based ligation using T4 RNA ligase 2 to ligate the first RNA molecule (5’ Oligo) to the second and third RNA molecules (reaction scheme on right hand side). Approach (3) was used to generate the data in Lane 2 of Figure 9B.

[0704] 69

[0705] 17228500 RGM1 RGM1FIG. 11 3’-5’ exonuclease activity of Exonuclease T (ExoT) and Ribonuclease R (RNase R) on the first and third RNA molecules (5’ Oligo and 3’ Oligo) which contain PS and 2’ O-Me modifications. The unprotected 3’ end of the first RNA molecule (5’ Oligo) means that the first RNA molecule (5’ Oligo) is susceptible to degradation by both ExoT and RNase R (left gel). The third RNA molecule (3’ Oligo) has a 5’ monophosphate (5’P) end and a modified 3’OH end and is susceptible to degradation by RNase R (left and middle gel) but is completely resistant to degradation by ExoT (left and right gel). RNase R is used at 1U / ug of oligos and ExoT is used at 15U / ug of oligos. The lengths of the first and third RNA molecules (5’ Oligo and 3’ Oligo) are indicated in the labels on the gels. A schematic of the first and third RNA molecules (5’ Oligo and 3’ Oligo) is provided on the right.

[0706] FIG. 125’-3’ exonuclease activity of XRN1 on the first and second RNA molecules (5’ Oligo and IVT RNA). The first RNA molecule (5’ Oligo) contains a 5’ OH end and PS and 2' O-Me modifications at the first 2 bases at the 5’ end. The first RNA molecule is resistant to XRN1 exonuclease activity (left gel). The second RNA molecule (IVT RNA) has a 5’ triphosphate (5’ppp) end and is resistant to XRN1 exonuclease activity (right gel, lane 2). When the second RNA molecule (IVT RNA) is pretreated via enzymatic dephosphorylation, which results in a 5’ monophosphate (5’P) end, the second RNA molecule (IVT RNA) is susceptible to XRN1 exonuclease activity (right gel, lane 3). The length of the first and second RNA molecules (5’ Oligo and IVT RNA) are indicated in the labels on the gels. A schematic of the first and second RNA molecules (5’ Oligo and IVT RNA) is provided on the right.

[0707] FIG. 13 Schematic of EcoToxNI based approach for sgRNA / pegRNA synthesis. EcoToxNI cleaves at a unique conserved GAA / AU site in the gRNA scaffold of an IVT product to generate the second RNA molecule (IVT RNA). This obviates the constraint of the first two nucleotides of the second RNA molecule being GG nucleotides due to the GG sequence being essential for T7 / SP6 polymerase based IVT. Subsequent enzymatic modification of the second RNA molecule (IVT RNA) allows for a ssDNA splint based approach for ligating the first, second and third RNA molecules using T4 RNA ligase 2 (reaction scheme on left hand side of Figure 13). An alternative approach is to ligate the first, second and third RNA molecules (5’ Oligo, IVT RNA and 3’ Oligo) in the presence of T4 RNA ligase 1 (reaction scheme on right hand side of Figure 13). A combination of the two approaches can be used which relies on sequential ligation of the third RNA molecule (3’ Oligo) to the second RNA molecule (IVT RNA) using RNA ligase 1 followed by ssDNA splint based ligation of the first RNA molecule (5’ Oligo) with T4 RNA ligase 2.

[0708] Fig. 14 EcoToxNI (ETN1) cleavage activity on sgRNA / pegRNA sequence. The RNA cleavage activity of EcoToxNI on an IVT generated RNA molecule of 124 nt was assessed at different timepoints. Cleavage of the IVT generated RNA molecule of 124 nt by EcoToxNI generates a second RNA molecule (IVT RNA) of 107 nt. The EcoToxNI was used at a concentration of 5U / ug of oligos at 37 °C. The optimal conditions for EcoToxNI cleavage are highlighted by a rectangle on the gel. These conditions (EcoToxNI at 5U / ug of oligos and incubated for 60 min at 37 °C) resulted in the majority of RNA being digested to produce the second RNA molecule (IVT RNA; 107 nt). A schematic of the IVT

[0709] 70

[0710] 17228500 RGM1 RGM1P262744WQ00

[0711] generated RNA molecule before digestion by EcoToxNI and the second RNA molecule produced after digestion by EcoToxNI is provided on the right.

[0712] Fig. 15 EcoToxNI based synthesis of pegRNA / sgRNA. The pegRNA / sgRNA is synthesized via the ssDNA splint ligation based approach illustrated in Figure 13. The pegRNA / sgRNA synthesised is 171 nt. The first RNA molecule (5’ Oligo; 35 nt) contains the spacer RNA sequence and part of the gRNA scaffold sequence and is modified at the 5’ end with PS and 2’0-Me modifications. The second RNA molecule (IVT RNA; 107 nt) is formed via digestion of the IVT generated RNA molecule (124 nt) with EcoToxNI (ETN1) and contains the gRNA scaffold and part of the RTT sequence. The third RNA molecule (3’ Oligo; 29 nt) contains part of the RTT sequence and the PBS sequence. The asterisk shows the synthesis of the ligated product in a non-purified sample (lane 3) as compared to the control of unmodified IVT RNA of the expected size (171 nt; lane 4).

[0713] EXAMPLES

[0714] Example 1

[0715] Nucleic acid synthesis: the following nucleic acids were chemically synthesized by Integrated DNA Technologies (IDT):

[0716] • an RNA oligonucleotide of 20 nucleotides encoding a spacer sequence (herein a first RNA molecule) and including phosphorothioated (PS) nucleotides at the 5’ end (SEQ ID NO: 1); • an RNA oligonucleotide of 16 nucleotide encoding a portion of the RTT and a primer binding site (PBS) (herein a third RNA molecule) and including phosphorothioated (PS) nucleotides at the 3’ end (SEQ ID NO: 3);

[0717] • a single stranded splint DNA (SEQ ID NO: 4); and

[0718] • a double stranded DNA to serve as a template (dsDNA template) directly or post PCR amplification for in vitro transcription (IVT) (SEQ ID NO: 5).

[0719] The sequences of these nucleic acids are provided in Table 1.

[0720] In vitro transcription (IVT) of the pegRNA scaffold region: IVT was performed using T7 RNA polymerase in a reaction mixture containing 1 x T7 RNA Polymerase Reaction Buffer and T7 RNA polymerase (T7 Flashscribe kit, Cellscript), 10 mM DTT, pyrophosphatase and 2.5 pg dsDNA template in a total volume of 50 pl. Nucleoside triphosphates (GTP, ATP, CTP, UTP) were supplemented to a final concentration of 10mM and the reaction mixture was incubated at 37 °C for 120 min. The dsDNA template was then enzymatically digested by adding 2 ul of RNase-Free DNase (T7 Flashscribe kit, Cellscript) at 37 °C for 15 min. The IVT RNA was purified with sodium acetate or ammonium acetate precipitation solution (Invitrogen). The IVT RNA (herein a second RNA molecule) is a 108 nucleotide RNA encoding a scaffold region and a first portion of a Reverse transcription template (RTT).

[0721] 71

[0722] 17228500 RGM1 RGM1Enzymatic treatment and ligation: The IVT RNA was dephosphorylated using 5’ Pyrophosphohydrolase (RPPH) (NEB) to leave a single 5'-monophosphate for the subsequent ligation reaction. For RNA ligation, 250 pmol each of the three RNA molecules, and an equimolar amount of the single stranded splint DNA, were added to 1 * annealing buffer (60mM NaCI, 6mM Hepes pH 7.5, 0.2mM MgCI2) in a total volume of 100 pl. This mixture was incubated for 2 min at 90 °C and then gradually cooled to room temperature. Subsequently, ligation was performed with 2 pl of 10* T4 RNA Ligase 2 buffer, T4 RNA Ligase 2 (NEB) as per manufacturer’s instructions, followed by incubation at 25 °C for 1 h. The splint DNA was digested with RNase-Free DNase (Cellscript) for 20 min at 37 °C. The ligation product was purified with sodium acetate precipitation solution and resuspended in nuclease free water.

[0723] Samples of the purified ligation product were analysed using 2% denaturing agarose gel electrophoresis (Figure 7) and 15% denaturing urea polyacrylamide gel electrophoresis (PAGE) (Figure 8). Figures 7 and 8 show that a high purity full length ligated pegRNA of 144 nucleotides was produced (see Ligated pegRNA 144 nt in Figures 7 and 8). The absence of unligated RNA components confirms a highly efficient splint ligation of the three RNA molecules.

[0724] Table 1

[0725]

[0726] 72

[0727] 17228500 RGM1 RGM1

[0728]

[0729] * indicates a phosphorothioated linkage between two nucleotides. For example, “G*G” means that the phosphorothioated linkage is between the two G nucleotides.

[0730] / 5phos / denotes a 5’-phosphate (i.e. a 5’ GMP).

[0731] / 5TriPhos / denotes 5’-triphosphate (i.e. a 5’ GTP)

[0732] “r” preceding A, C, G or U denotes a ribonucleotide.

[0733] Example 2

[0734] Nucleic acid synthesis: the following nucleic acids were chemically synthesized by IDT or Genscript:

[0735] • an RNA oligonucleotide of 19 nucleotides encoding a spacer sequence (5’ Oligo, herein a first RNA molecule) and including phosphorothioated (PS) and 2’-O-methylation (2’ O-Me) modified nucleotides at the 5’ end (IDT);

[0736] 73

[0737] 17228500 RGM1 RGM1• an RNA oligonucleotide of 20 nucleotides encoding a portion of the MS2 stem loop to mediate interaction of an RNA aptamer to MCP (MS2 Coat protein) tagged base editors (3’ Oligo, herein a third RNA molecule) and including phosphorothioated (PS) and 2’-O-methylation (2’ O-Me) modified nucleotides at the 3’ end (IDT);

[0738] • an RNA oligonucleotide of 89 nt which encodes a scaffold region and a first portion of a MS2 stem loop sequence (herein a second RNA molecule) (Genscript);

[0739] • an RNA oligonucleotide of 128 nt which includes the sequences of the first RNA molecule, the second RNA molecule and the third RNA molecule and includes phosphorothioated (PS) and 2’-O-methylation (2’ O-Me) modified nucleotides at the 5’ and 3’ ends (Genscript);

[0740] • a single stranded splint DNA (IDT); and

[0741] • a double stranded DNA (IDT) to serve as a template (dsDNA template) directly or post PCR amplification for in vitro transcription (IVT).

[0742] The sequences of the above nucleic acids are provided in Table 2.

[0743] In vitro transcription (IVT) to generate the second RNA molecule (IVT RNA; 89 nt) encoding the scaffold region and part of the MS2 stem loop: IVT was performed using T7 RNA polymerase in a reaction mixture containing 1 x T7 RNA Polymerase Reaction Buffer and T7 RNA polymerase (T7 Flashscribe kit, Cellscript), 10 mM DTT, pyrophosphatase and 2.5 pg dsDNA template in a total volume of 50 pl. Nucleoside triphosphates (GTP, ATP, CTP, UTP) were supplemented to a final concentration of 10mM and the reaction mixture was incubated at 37 °C for 240 min. The dsDNA template was then enzymatically digested by adding 2 ul of RNase-Free DNase (T7 Flashscribe kit, Cellscript) at 37 °C for 15 min. The IVT RNA was purified with sodium acetate or ammonium acetate precipitation solution (Invitrogen) or a Monarch Spin RNA Cleanup Kit.

[0744] The following sgRNA / pegRNA products were generated using the chemically synthesized and IVT generated RNA molecules outlined above (Figure 9B, 9C):

[0745] • sgRNA / pegRNA comprising chemically synthesized first and third RNA molecules (5’ Oligo of 19 nt and 3’ Oligo of 20 nt) and IVT synthesized second RNA molecule (IVT RNA of 89 nt) ligated together, wherein phosphorothioated (PS) and 2’-O-methylation (2’ O-Me) modified nucleotides are present at the 5’ end and the 3’ end of the sgRNA / pegRNA (lane 2);

[0746] • sgRNA / pegRNA comprising chemically synthesized first and third RNA molecules (5’ Oligo of 19 nt and 3’ Oligo of 20 nt) and chemically synthesized second RNA molecule (SPOS RNA of 89 nt) ligated together, wherein phosphorothioated (PS) and 2’-O-methylation (2’ O-Me) modified nucleotides are present at the 5’ end and the 3’ end of the sgRNA / pegRNA (lane 3); and

[0747] • sgRNA / pegRNA comprising a full length chemically synthesized sgRNA / pegRNA of 128 nt which includes the sequences of the first, second and third RNA molecules as one chemically

[0748] 74

[0749] 17228500 RGM1 RGM1synthesized molecule wherein phosphorothioated (PS) and 2’-O-methylation (2’ O-Me) modified nucleotides are present at the 5’ end and the 3’ end of the sgRNA / pegRNA (lane 4).

[0750] Enzymatic treatment of the second RNA molecule and ligation to generate sgRNA / pegRNA comprising chemically synthesized first and third RNA molecules (5’ Oligo and 3’ Oligo) and an IVT generated second RNA molecule (IVT RNA): IVT RNA (IVT RNA; 89 nt) was preheated in the presence of 3’ Oligo at (1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :10) molar ratios. For example, 4 ug of IVT RNA was mixed with 5.4 ug of 20 nt 3’ Oligo (1 :6 molar ratio) in the presence of 42% formamide (or in the range of 10%-80%), heated at 80 °C (or in the range of 50-90 °C) for 5 mins (or in the range of 2-30 mins) and then snap cooled to 4 °C on ice. The IVT RNA and the 3’ Oligo in the preheated RNA mixture were ligated using the ssRNA ligase T4 RNA ligase 1 (NEB or MCLAB) as per manufacturer’s guidelines. A 60 ul reaction containing 9.4 ug preheated RNA, 1x T4 RNA ligase 1 buffer, ATP (1-5 mM), 10 mM MgCI2, PEG8000 (10-25%), RNase inhibitor (1 U / ul), T4 RNA ligase 1 (40 unit) was incubated at 25 °C for 2-4 hours. This reaction was followed by purification using the Monarch Spin clean up kit (alternatively salt-based precipitation could be used, or the reaction could be directly used for RppH treatment).

[0751] Removal of pyrophosphate from the 5’ triphosphate (5’ppp) at the 5’ end of the ligated second and third RNA molecules (IVT RNA + 3’ Oligo) to generate a 5’ monophosphate end is carried out with RNA 5’ Pyrophosphohydrolase (RppH) treatment using standard manufacturer’s guidelines. This RppH treatment is carried out to enable further ligation to the first RNA molecule (5’ Oligo). A typical 100 ul reaction containing 10 ul 10 x NEB buffer 2.0, 25 ug of ligated IVT RNA + 3’ Oligo, 5 ul (25 Units) of RppH is incubated at 37 °C for 1-2 hour, and is followed by Column Clean up or precipitation-based purification. Alternatively, 5’ triphosphate (5’ppp) at the 5’ end of the IVT RNA can be first converted to 5’OH using a phosphatase such as Calf intestinal alkaline phosphatase (CIAP) and then treated by T4 polynucleotide kinase to produce a second RNA molecule with a 5’ monophosphate end that can be ligated to the first RNA molecule.

[0752] Equimolar amounts of ligated 5’ monophosphate IVT RNA+3’ Oligo (109 nt): 5’ Oligo (19 nt): ssDNA (128 nt) (100-500 pmol range) is heated at 95 °C (or in the range of 65-95 °C) for 2-10 mins in presence of 1x annealing buffer (60mM NaCI, 6mM Hepes pH 7.5, 0.2mM MgCI2) followed by slow cooling to room temperature (RT) over about 3 hours to form a RNA:DNA hybrid complex. Following hybridization, the first RNA molecule (5’ Oligo) is ligated to the 5’-monophosphate IVT RNA + 3’ Oligo on the RNA:DNA hybrid complex using T4 RNA Ligase 2 (dsRNA ligase) (NEB, MCLAB) as per manufacturer’s guidelines, at an enzyme amount of approximately 25 units per 10 pg of RNA:DNA hybrid in a reaction volume of about 20 pL, with incubation at approximately 25 °C (range 25-37 °C) for about 2 (1 -3 hours) hours. Ligated full length sgRNA / pegRNA is released by DNAase I or duplex DNase treatment for 30 mins and this is followed by column clean up or precipitation steps. The ligation product is subsequently analysed by denaturing polyacrylamide gel electrophoresis using a 10% urea gel.

[0753] 75

[0754] 17228500 RGM1 RGM1Ligation reaction to generate sgRNA / pegRNA comprising chemically synthesized first, second and third RNA molecules:

[0755] In this experiment, instead of the second RNA molecule being produced via in vitro transcription, the second RNA molecule of 89 nt was chemically synthesised by solid phase oligonucleotide synthesis (SPOS) by Genscript. The chemically synthesized second RNA molecule (SPOS RNA) has a 5’-monophosphate end. Using a similar approach as outlined above, the SPOS RNA is first preheated with the 3’ Oligo (1 :6 molar ratio). This is followed by ligation using ssRNA ligase (T4 RNA ligase 1) and column or precipitation-based purification. SPOS RNA + 3’ Oligo is hybridised to the 5’ Oligo and a ssDNA splint at equimolar ratios by heating and slow cooling in the presence of 1x annealing buffer (as outlined above) to generate a RNA:DNA hybrid complex.

[0756] Following hybridization, the first RNA molecule (5’ Oligo) is ligated to the 5’-monophosphate SPOS RNA + 3’ Oligo on a RNA:DNA hybrid using T4 RNA Ligase 2 (dsRNA ligase) (NEB, MCLAB) as per manufacturer’s guidelines, at an enzyme amount of approximately 25 units per 10 pg of RNA:DNA hybrid in a reaction volume of about 20 pL, with incubation at approximately 25 °C (range 25-37 °C) for about 2 (1-3 hours) hours. Ligated full length sgRNA / pegRNA is released by DNAase I or duplex DNase treatment for 30 mins (as per manufacturer’s guidelines) and this followed by purifying by column clean up or precipitation steps. The ligation product is subsequently analyzed by denaturing polyacrylamide gel electrophoresis using a 10% urea gel.

[0757] Samples of the purified full length ligation products which differ in the synthesis of second RNA molecule i.e. by IVT (IVT RNA, lane 2) or solid phase chemical synthesis (SPOS RNA, lane 3), and the fully chemically synthesized RNA molecule (lane 4) of 128 nt were analysed using 10% denaturing urea polyacrylamide gel electrophoresis (PAGE) (Figure 9B). Schematic illustrations of the sgRNA / pegRNA molecules generated are shown in Figure 9C. The percentage of full length (128 nt) sgRNA / pegRNA was analysed via densitometric analysis (Figure 9D). Figures 9B and 9D show that a much greater purity of sgRNA / pegRNA product is obtained when the second RNA molecule is synthesized via IVT (IVT RNA; 89 nt) (lane 2) compared to chemical synthesis of the second RNA molecule (SPOS RNA; 89 nt) (lane 3). The purity of the sgRNA / pegRNA molecule where the first, second and third RNA molecules are chemically synthesized and ligated together (lane 3) is comparable to the yield of sgRNA / pegRNA when the whole 128 nt molecule is chemically synthesized (lane 4).

[0758] This data shows that an approach using IVT for synthesis of the longer second RNA molecule (shown for examples of 89 nt and above) is beneficial for obtaining a higher purity and better quality sgRNA / pegRNA over an approach using only chemically synthesised RNA molecules for generating long sgRNA / pegRNA molecules of 120 nt and above. Combining a long second RNA molecule generated via IVT (80 nt and above) with chemically synthesised and modified shorter 5’ and 3’ Oligos (below 40 nt) has benefits in terms of high purity, enzymatic scalability and environmental sustainability due to toxic waste reduction.

[0759] 76

[0760] 17228500 RGM1 RGM1Table 2: 128 nt base editing sgRNA / pegRNA

[0761]

[0762] 77

[0763] 17228500 RGM1 RGM1

[0764]

[0765] * indicates a phosphorothioated linkage between two nucleotides. For example, “G*G” means that the phosphorothioated linkage is between the two G nucleotides.

[0766] m indicates 2’0-methylated nucleotides

[0767] / 5phos / denotes a 5’-phosphate (i.e. a 5’ GMP).

[0768] / 5TriPhos / denotes 5’-triphosphate (i.e. a 5’ GTP)

[0769] “r” preceding A, C, G or U denotes a ribonucleotide.

[0770] Example 3

[0771] 3’-5’ exonuclease activity of Exonuclease T (ExoT) and Ribonuclease R (RNase R) on modified first and third RNA molecules (5’ Oligo and 3’ Oligo): The susceptibility of unprotected 3’ OH ends of the PS-2'OMe modified first RNA molecule (5’ Oligo) to exonuclease degradation was assessed with both ExoT and RNase R (gel on the left). The third RNA molecule (3’ Oligo) of 20 nt with a protected 3’OH end (PS-2’OMe modifications) and first RNA molecule (5’ Oligo) of 19 nt with an unprotected 3’OH end were assessed for 3’-5’ exonuclease degradation using ExoT (15 U (5-50 U range) per ug of oligos at 25 °C for 1 hr in presence of 1X NEBuffer 4 (NEB)) and RNase R (1 U (1-5 U range) per ug of oligos at 37 C for 1 hr in presence of 1X RNase R buffer (NEB)) followed by reaction termination with about 10 mM EDTA as per manufacturer’s guidelines. Control reactions are performed under identical conditions in the absence of enzyme. Following exonuclease treatment, RNA products were analyzed by denaturing polyacrylamide gel electrophoresis using a 10% urea gel. Two different length third RNA molecules were used in this exonuclease degradation study i.e. 20 nt and 29 nt third RNA molecules (3’ Oligos) with PS-2'OMe modified 3’OH ends were assessed using identical conditions. These experiments confirm that the third RNA molecule (3’ Oligo) carrying PS-2'OMe modified 3’OH ends is susceptible to some digestion by RNase R (Figure 11 , left and middle gel) but is completely resistant to ExoT treatment (Figure 11 , left and right gel). A schematic of the activity of the exonucleases used is provided on the right side of Figure 11.

[0772] 5’-3’ exonuclease activity of XRN1 on modified first RNA molecules (5’ Oligos) and second RNA molecules (IVT RNA): First RNA molecules (5’ Oligos) of 35 and 51 nt with protected 5’OH ends (PS-2’OMe) were assessed for 5’-3’ exonuclease degradation using XRN1 (1 U (1-4 U range) per ug of oligos at 37 °C for 1 hr in presence of 1X NEBuffer 3 (NEB)) followed by reaction termination with 10 mM EDTA as per manufacturer’s guidelines. Control reactions are performed under identical conditions in the absence of enzyme. First RNA molecules (5’ Oligos) with modified (PS-2’OMe) 5’OH ends are resistant against XRN1 exonuclease activity (Figure 12, left gel). XRN1 was used to test the susceptibility of the second RNA molecule (IVT RNA) of 89 nt to exonuclease degradation when the second RNA molecule had a 5’ triphosphate (5’ppp) end and also when the second RNA molecule was treated with RppH to give a 5’-monophosphate (5’P) end. The experiments were carried out using identical conditions. While the second RNA molecule (IVT RNA) with a 5’triphosphate (5’ppp) end is resistant to XRN1 exonuclease activity (Figure 12, right gel, lane 2), the second RNA molecule (IVT

[0773] 78

[0774] 17228500 RGM1 RGM1RNA) with a 5’-monophosphate (5’P) end is susceptible to XRN1 activity (Figure 12, right gel, lane 3). The length of the first RNA molecule (5’ Oligo) and the second RNA molecule (IVT RNA) tested are indicated in the labels of the gels in Figure 12. A schematic of the activity of XRN1 on the first RNA molecule (5’ Oligo), or the second RNA molecule (IVT RNA) with either a 5’triphosphate (5’ppp) or 5’-monophosphate (5’P) end is provided on the right side of Figure 12. This data clearly validates the use of3’-5’ exonucleases and 5’-3’ exonuclease in enzymatic degradation of intermediate RNA products (5’ and 3’ Oligos and 5’-monophosphate and 5’ triphosphate (5’ppp) IVT RNA) to improve the purity of ligated RNA product.

[0775] Example 4

[0776] Nucleic acid synthesis: the following nucleic acids were chemically synthesized:

[0777] • an RNA oligonucleotide of 35 nucleotides encoding a spacer sequence (herein a first RNA molecule) and including phosphorothioated (PS) and 2’-O-methylation (2’0Me) nucleotides at the 5’ end (5’ Oligo);

[0778] • an RNA oligonucleotide of 29 nucleotides encoding a portion of the RTT and a primer binding site (PBS) (herein a third RNA molecule) and including phosphorothioated (PS) and 2’-O- methyation (2’0Me) nucleotides at the 3’ end (3’ Oligo);

[0779] • a single stranded splint DNA; and

[0780] • a double stranded DNA to serve as a template (dsDNA template) directly or post PCR amplification for in vitro transcription (IVT).

[0781] The sequences of these nucleic acids are provided in Table 3.

[0782] In vitro transcription (IVT) of the sgRNA / pegRNA encoding the scaffold region and part of a reverse transcription template (RTT): IVT was performed using T7 RNA polymerase in a reaction mixture containing 1* T7 RNA Polymerase Reaction Buffer and T7 RNA polymerase (T7 Flashscribe kit, Cellscript), 10 mM DTT, pyrophosphatase and 2.5 pg dsDNA template in a total volume of 50 pl. Nucleoside triphosphates (GTP, ATP, CTP, UTP) were supplemented to a final concentration of 10mM and the reaction mixture was incubated at 37 °C for 240 min. The dsDNA template was then enzymatically digested by adding 2 ul of RNase-Free DNase (T7 Flashscribe kit, Cellscript) at 37 °C for 15 min. The IVT RNA was purified with sodium acetate or ammonium acetate precipitation solution (Invitrogen) or a Monarch Spin RNA Cleanup Kit. The IVT RNA (herein a second RNA molecule) is a 124 nucleotide RNA encoding a scaffold region and a first portion of a RTT.

[0783] The following products were generated using IVT generated RNA molecules (Figures 14 and 15):

[0784] • IVT synthesized RNA molecule (herein a second RNA molecule) of 124 nt which is cleaved with endoribonuclease EcoToxNI (ETN1) at GAA / AU site resulting in a 107 nt RNA molecule (IVT RNA) with a 5’OH end;

[0785] 79

[0786] 17228500 RGM1 RGM1• pegRNA of 171 nt comprising chemically synthesized first and third RNA molecules (5’ Oligo of 35 nt and 3’ Oligo of 29 nt) ligated to a second RNA molecule (IVT RNA of 107 nt), wherein phosphorothioated (PS) and 2’-O-methylation (2’OMe) nucleotides are present at the 5’ end and the 3’ end of the pegRNA; and

[0787] • IVT generated RNA molecule of 171 nt which does not have nucleotide modifications and includes the sequences of the first, second and third RNA molecules.

[0788] EcoToxNI approach for generating the second RNA molecule (for ligation): In this experiment, a second RNA molecule of 124 nt generated by IVT (approximately 100 pmol, corresponding to about 4 pg) is digested using an endoribonuclease (ETN1) at an enzyme amount of approximately 20 units in a reaction buffer comprising about 25 mM Tris-CI and 25 mM NaCI, wherein the enzyme is diluted in a buffer comprising about 10 mM Tris-CI and 50 mM NaCI. The reaction is incubated at approximately 37 °C for about 1 hour, followed by enzyme inactivation at approximately 60 °C for about 20 minutes. The second RNA molecule (IVT RNA) cleaved by EcoToxNI is purified using a silica-based column cleanup method (Monarch cleanup spin column) and analyzed by 10% denaturing urea polyacrylamide gel electrophoresis. In this example, ETN1 recognizes an AAA.AU sequence motif and cleaves the second RNA molecule (generated by IVT) after the third adenosine residue to generate an RNA molecule (a second RNA molecule) having a 5'-hydroxyl terminus of AU. The 5’ end of the second RNA molecule is subsequently converted to a 5'-phosphate by incubating approximately 20 pg of the second RNA molecule with about 40 units of T4 polynucleotide kinase (T4 PNK) for approximately 1 hour under conditions sufficient to produce a ligatable second RNA molecule.

[0789] Further, the T4 polynucleotide kinase-treated second RNA molecule is hybridized to a single-stranded DNA splint complementary thereto, wherein first and third RNA molecules, which are each shorter than the kinase-treated second RNA molecule and are chemically synthesized with terminal modifications, are also complementary to the same DNA splint. The RNA fragments and DNA splint are combined in equimolar amounts of approximately 250 pmol, heated to about 95 °C for approximately 2 minutes, and gradually cooled to room temperature over about 3 hours to form an RNA:DNA hybrid complex. Following hybridization, the first and third RNA molecules are ligated to the kinase-treated second RNA molecule using T4 RNA ligase 2 at an enzyme amount of approximately 25 units per 10 pg of RNA:DNA hybrid in a reaction volume of about 20 pL, with incubation at approximately 25 °C for about 2 hours. The ligation product is subsequently analyzed by denaturing polyacrylamide gel electrophoresis using a 10% urea gel.

[0790] Table 3: EcoToxNI Strategy for pegRNA / sgRNA synthesis

[0791]

[0792] 80

[0793] 17228500 RGM1 RGM1

[0794]

[0795] 81

[0796] 17228500 RGM1 RGM1CLAUSES

[0797] 1. A method for producing a guide RNA, wherein the method comprises:

[0798] a) providing a first RNA molecule, a second RNA molecule, and a third RNA molecule, wherein the first RNA molecule comprises a nuclease-resistant nucleotide and the third RNA molecule comprises a nuclease-resistant nucleotide; and

[0799] b) ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule, wherein the ligation is performed by splint ligation;

[0800] wherein the guide RNA comprises, in the 5’-3’ direction, a spacer sequence and a scaffold region; and

[0801] wherein the first RNA molecule is a chemically synthesized RNA molecule, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule, and the third RNA molecule is a chemically synthesized RNA molecule.

[0802] 2. The method of clause 1 , wherein the first RNA comprises at least a portion of the spacer sequence and the second RNA molecule comprises at least a portion of the scaffold region.

[0803] 3. The method of clause 1 or clause 2, wherein the guide RNA is a prime editing guide RNA (pegRNA) and wherein the pegRNA comprises, in the 5’-3’ direction, a spacer sequence, a scaffold region, a reverse transcription template (RTT) and a primer binding site (PBS).

[0804] 4. The method of clause 3, wherein either:

[0805] g) the second RNA molecule comprises the reverse transcription template (RTT);

[0806] h) the third RNA molecule comprises the reverse transcription template (RTT); or

[0807] i) the second RNA molecule comprises a portion of the reverse transcription template (RTT) and the third RNA molecule comprises a portion of the reverse transcription template (RTT).

[0808] 5. The method of clause 3 or clause 4, wherein the third RNA molecule comprises the PBS.

[0809] 6. The method of any one of clauses 1-5, wherein step (a) comprises: producing the first RNA molecule and the third RNA molecule by chemical synthesis; and producing the second RNA molecule by in vitro transcription (IVT), optionally wherein the chemical synthesis comprises solidphase synthesis.

[0810] 7. The method of any one of clauses 1-6, wherein the second RNA molecule has a 5’-terminal GTP and step (a) comprises treating the second RNA molecule with a phosphatase to cleave phosphate groups from the 5’-terminal GTP to produce a second RNA molecule with a 5’-terminal GMP.

[0811] 82

[0812] 17228500 RGM1 RGM18. The method of any one of clauses 1-7, wherein step (b) comprises: annealing the first, second and third RNA molecules to a splint DNA; and ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule; optionally wherein the splint DNA is a single splint DNA molecule.

[0813] 9. The method of clause 8, wherein the splint DNA is a protected DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the protected DNA comprises x nuclease-resistant nucleotides at the 5’ end of the ssDNA cassette or 5’ of the ssDNA cassette, and y nuclease-resistant nucleotides at the 3’-end of the ssDNA cassette or 3’ of the ssDNA cassette, wherein x is at least 1 and y is at least 1.

[0814] 10. The method of clause 8, wherein the splint DNA is a protected DNA comprising a single-stranded DNA (ssDNA) cassette, and wherein the method comprises:

[0815] (a) providing a partially protected double-stranded DNA (dsDNA) comprising a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises a cassette, x nuclease-resistant nucleotides at the 5’ end of the cassette or 5’ of the cassette, and y nuclease-resistant nucleotides at the 3’-end of the cassette or 3’ of the cassette, wherein x is at least 1 and y is at least 1 ; and

[0816] (b) digesting the second strand of the partially protected dsDNA with an exonuclease thereby generating the protected DNA.

[0817] 11. The method of any one of clauses 8-10, wherein the splint DNA comprises a sequence comprising regions complementary to the scaffold region and spacer sequence.

[0818] 12. The method of any one of clauses 8-11 , wherein the method further comprises (c) digesting the splint DNA using DNase.

[0819] 13. The method of any one of clauses 1-12, wherein step (b) is performed using T4 RNA ligase.

[0820] 14. The method of any one of clauses 1-13, wherein the first RNA molecule comprises a nuclease- resistant nucleotide 5’ of the spacer sequence and / or wherein the third RNA molecule comprises a nuclease-resistant nucleotide.

[0821] 15. A guide RNA for use in a method of treating a disease, wherein the guide RNA is produced according to the method of any one of clauses 1-14 and wherein the guide RNA is used to edit a target gene by gene editing and thereby treat the disease.

[0822] 83

[0823] 17228500 RGM1 RGM1

Claims

CLAIMS1. A method for producing a guide RNA, wherein the method comprises:a) providing a first RNA molecule, a second RNA molecule, and a third RNA molecule, wherein the first RNA molecule comprises a nuclease-resistant nucleotide and the third RNA molecule comprises a nuclease-resistant nucleotide; andb) ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule; wherein the guide RNA comprises, in the 5’-3’ direction, a spacer sequence and a scaffold region; andwherein the first RNA molecule is a chemically synthesized RNA molecule, the second RNA molecule is an in vitro transcription (IVT) produced RNA molecule, and the third RNA molecule is a chemically synthesized RNA molecule.

2. The method of claim 1 , wherein step (b) comprises: annealing the first, second and third RNA molecules to a splint DNA; and ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule by splint ligation.

3. The method of claim 1 , wherein step (b) comprises: ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule to generate an intermediate ligation product; annealing the first RNA molecule and the intermediate ligation product to a splint DNA; and ligating the 3’ end of the first RNA molecule to the 5’ end of the intermediate ligation product.

4. The method of claim 2 or claim 3, wherein the splint DNA is a single splint DNA molecule.

5. The method of any one of claims 1-4, wherein step (b) is performed using T4 RNA ligase.

6. The method of claim 1 , wherein step (b) comprises: ligating the 3’ end of the first RNA molecule to the 5’ end of the second RNA molecule and ligating the 3’ end of the second RNA molecule to the 5’ end of the third RNA molecule by an RNA ligase.

7. The method of claim 6, wherein the RNA ligase is T4 RNA ligase 1 .

8. The method any one of claims 1-7, wherein the first RNA comprises at least a portion of the spacer sequence and the second RNA molecule comprises at least a portion of the scaffold region.

9. The method of any one of claims 1-8, wherein the guide RNA is a prime editing guide RNA (pegRNA) and wherein the pegRNA comprises, in the 5’-3’ direction, a spacer sequence, a scaffold region, a reverse transcription template (RTT) and a primer binding site (PBS).8417228500 RGM1 RGM110. The method of claim 9, wherein either:a) the second RNA molecule comprises the reverse transcription template (RTT);b) the third RNA molecule comprises the reverse transcription template (RTT); orc) the second RNA molecule comprises a portion of the reverse transcription template (RTT) and the third RNA molecule comprises a portion of the reverse transcription template (RTT).

11. The method of claim 9 or claim 10, wherein the third RNA molecule comprises the PBS.

12. The method of any one of claims 1-11 , wherein step (a) comprises: producing the first RNA molecule and the third RNA molecule by chemical synthesis; and producing the second RNA molecule by in vitro transcription (IVT), optionally wherein the chemical synthesis comprises solidphase synthesis.

13. The method of any one of claims 2-5 or 8-12, wherein the splint DNA is a protected DNA comprising a single-stranded DNA (ssDNA) cassette, wherein the protected DNA comprises x nuclease- resistant nucleotides at the 5’ end of the ssDNA cassette or 5’ of the ssDNA cassette, and y nuclease-resistant nucleotides at the 3’-end of the ssDNA cassette or 3’ of the ssDNA cassette, wherein x is at least 1 and y is at least 1 .

14. The method of any one of claims 2-5 or 8-12, wherein the splint DNA is a protected DNA comprising a single-stranded DNA (ssDNA) cassette, and wherein the method comprises:(a) providing a partially protected double-stranded DNA (dsDNA) comprising a first strand and a second strand, wherein the first strand of the partially protected dsDNA comprises a cassette, x nuclease-resistant nucleotides at the 5’ end of the cassette or 5’ of the cassette, and y nuclease-resistant nucleotides at the 3’-end of the cassette or 3’ of the cassette, wherein x is at least 1 and y is at least 1 ; and(b) digesting the second strand of the partially protected dsDNA with an exonuclease thereby generating the protected DNA.

15. A guide RNA for use in a method of treating a disease, wherein the guide RNA is produced according to the method of any one of claims 1-14 and wherein the guide RNA is used to edit a target gene by gene editing and thereby treat the disease.8517228500 RGM1 RGM1

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