Single guide RNA synthesis

Abstract

The present invention relates to methods for synthesizing sgRNA, in particular to large scale methods for synthesizing sgRNA.

Description

[0001] Single guide RNA synthesis

[0002] The present invention relates to a method for the synthesis of single guide RNAs (sgRNA) and in particular to large scale methods for the production of sgRNAs.

[0003] Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated enzyme (Cas) is a naturally occurring genome editing tool adopted from the prokaryotic adaptive immune defense system. Currently, CRISPR / Cas9-based genome editing has been becoming one of the most promising tools for treating human genetic diseases, including cardiovascular diseases, neurodisorders, and cancers. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual transactivating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure of a guide RNA (gRNA).

[0004] CRISP R / Cas9-based gene therapy has been extensively studied in preclinical and clinical treatments. Available approaches for the synthesis of gRNA include intracellular transcription of an exogenous plasmid and solid-phase synthesis using phosphoramidite chemistry.

[0005] There is a need for efficient and high yielding methods for synthesizing highly pure gRNA and sgRNA both in the small and large scale.

[0006] In a first aspect the present invention provides a method of synthesizing a single guide RNA (sgRNA) comprising: providing a first single stranded RNA fragment comprising a terminal region with a 3'-hydroxyl group and a second single stranded RNA fragment comprising a terminal region with a 5' phosphate moiety, wherein the first and second RNA fragments have a length of 45 - 51 nucleotides (nt), providing a single stranded DNA oligonucleotide having a length from 18 - 28 nt, wherein the DNA oligonucleotide comprises a first region being complementary to the first RNA fragment at its 3 '-hydroxyl group end and a second region being complementary to the second RNA fragment at its 5 '-phosphate moiety end, wherein each of the two complementary regions of the DNA oligonucleotide have at least a length of 9 nucleotides.

[0007] Hybridizing the two RNA fragments and the DNA oligonucleotide to form a complex and ligating the first and the second RNA fragment at a ligation site present between the two RNA fragments using a ligase to synthesize the sgRNA.

[0008] In an embodiment of the method, the sgRNA has a length of 100 nt.

[0009] In an embodiment of the method, the first RNA fragment has a length of 49 nt and the second RNA fragment has a length of 51 nt or the first RNA fragment has a length of 51 nucleotides and the second RNA fragment has a length of 49 nt.

[0010] In an embodiment of the method, the two complementary regions of the DNA oligonucleotide have the same length.

[0011] In an embodiment of the method, the DNA oligonucleotide has a length of 26 nt and each of the two complementary regions has a length of 13 nt.

[0012] In an embodiment of the method, the DNA oligonucleotide has a length of 28 nt and each of the two complementary regions has a length of 14 nt. In an embodiment of the method, the two complementary regions of the DNA oligonucleotide are 100% complementary to the respective RNA fragment regions.

[0013] In an embodiment of the method, the sgRNA comprises a crRNA segment and tracrRNA segment.

[0014] In an embodiment of the method, the ligation site lies within the tracrRNA segment.

[0015] In a second aspect, the present invention provides a reaction mixture for the large-scale synthesis of sgRNA comprising:

[0016] 0.05 - 0.2 rnM single stranded oligonucleotides as defined above,

[0017] 0.05 - 0.2 rnM single stranded DNA oligonucleotide as defined above,

[0018] 0.001 mg / ml - 0.5 mg / ml ligase, in an aqueous solution at about pH 7.0, wherein the reaction mixture has a volume of at least 10'5liters to 10000 liters.

[0019] In an embodiment of the reaction mixture, the volume is at least 1 to 5000 liters, preferably 5 to 2500 liters, preferably 10 to 1000 liters.

[0020] In an embodiment of the reaction mixture, the reaction mixture further comprises the additives acetonitrile, ATP, DTT, PEG, magnesium or manganese and DMSO.

[0021] In an embodiment of the reaction mixture, the ligase is a RNA ligase, preferably a T4 RNA ligase 2.

[0022] In an embodiment of the reaction mixtures, the enzyme was immobilized on agarose beads to ease separation of the ligation products and reuse of the enzyme for subsequent reactions.

[0023] In an embodiment of the reaction mixture,, the reaction mixture comprises:

[0024] 10 / zg to 50g single stranded oligonucleotides,

[0025] 10 / zg to 50g single stranded DNA oligonucleotides

[0026] 1 / zg to 200mg ligase in a volume of 20 / zl 7 liters.

[0027] Definitions

[0028] As used herein, the term “guide RNA” or “gRNA” refers to a site-specific targeting RNA that can bind an RNA-guided endonuclease to form a complex, and direct the activities of the bound RNA- guided endonuclease (such as a Cas endonuclease) to a specific target sequence within a target nucleic acid. The gRNA is a duplex RNA made up of two parts: crisprRNA (crRNA), a nucleotide sequence complementary to the target DNA, and a tracrRNA, which serves as a binding scaffold for the Cas nuclease, wherein the crRNA and tracrRNA hybridize to each other to form a duplex.

[0029] As used herein, a single guide RNA (sgRNA) refers to a guide RNA which comprises the CRISPR RNA and the tracer RNA in one molecule.

[0030] As used herein, the term “Cas endonuclease” or “Cas nuclease” refers to an RNA-guided DNA endonuclease associated with the CRISPR adaptive immunity system.

[0031] Unless otherwise indicated “nuclease” and “endonuclease” are used interchangeably herein to refer to an enzyme which possesses endonucleolytic catalytic activity for polynucleotide cleavage. As used herein, the term “cleavage” refers to the breakage of the covalent backbone of a DNA molecule. The cleavage can be a single-stranded cleavage or a double-stranded cleavage. For example, a double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events.

[0032] As used herein, the term “hybridizing” or “hybridize” refers to the pairing of substantially complementary or complementary nucleic acid sequences within two different strands. Pairing can be achieved by any process in which a nucleic acid sequence joins with a substantially or fully complementary sequence through base pairing to form a hybridization complex. “Hybridizing” or “hybridize” can comprise denaturing the nucleic acid sequences to disrupt the inter- or intra- molecular structure(s) (e.g., secondary structure(s)) in the molecule.

[0033] Short description of the figures:

[0034] Fig. 1A shows the High-Performance Liquid Chromatography (HPLC) chromatogram of the ligation reaction using a 18 mer DNA adaptor and Fig. IB shows the Liquid Chromatography -Mass Spectrometry (LC-MS) chromatogram of the ligation reaction using a 18 mer DNA adaptor.

[0035] Fig 2A shows the HPLC chromatogram of the ligation reaction using a 16 mer DNA adaptor and Fig. 2B shows the LC-MS chromatogram of the ligation reaction using a 16 mer DNA adaptor.

[0036] Fig. 3 shows the ligation conversion (expressed in %), over time (expressed in hours) as a function of the ligation site.

[0037] Fig. 4 shows the HPLC chromatogram of the purified sgRNA A

[0038] Fig. 5 shows the LC-MS analysis of the purified sgRNA A

[0039] Fig. 6 shows the HPLC chromatogram of the purified sgRNA B

[0040] Fig. 7 shows the LC-MS analysis of the purified sgRNA B

[0041] Fig, 8 shows the graphical representation of the functionalization of a cyanogen bromide-activated matrix.

[0042] Fig. 9 shows the conversion (expressed in %) to product, over time (expressed in hours) of the ligations using two differently stored batches of immobilized enzyme.

[0043] Fig. 10 shows the LC-MS chromatograms of the reaction crude from a 2h ligation using the immobilized enzyme stored in solution respectively on the 1st day (Fig. 10A) and 4th day (Fig. 10B).

[0044] Fig. 11 shows the LC-MS chromatograms of the reaction crude from a 2h ligation using the immobilized enzyme stored as lyophilized solid respectively on the 1st day (Fig. HA) and 4th day (Fig. 11B).

[0045] Fig. 12 shows the variation in the conversion to products when the enzyme loading is increased or decreased by 10%.

[0046] Fig. 13 shows the variation in the conversion to products when the enzyme loading is increased or decreased by 50%.

[0047] Fig. 14 shows the variation in the conversion to products when the acetonitrile content is increased up to 15%.

[0048] Examples: This invention deals mainly with conventional techniques of molecular biology, biochemistry and nucleic acid chemistry, which are known to those skilled in the art.

[0049] • Example 1 : Investigation of the ligation efficiency with adaptors of different length

[0050] • Example 2: Investigation of the ligation efficiency as a function of the ligation site

[0051] • Example 3: Synthesis and purification of sgRNA A and B via ligation (600 mg scale)

[0052] • Example 4: Multigram synthesis and purification of sgRNA A and B

[0053] • Example 5: Ligation with immobilized T4-Ligase on covalent support (Agarose CN- bromide)

[0054] • Example 6: Investigation of the ligation with different quantities of enzyme

[0055] • Example 7: Effect of sgRNA fragment and splint concentrations on ligation

[0056] • Example 8: Buffer composition

[0057] • Example 9: Buffer optimization via additives

[0058] Example 1: Investigation of the ligation efficiency with adaptors of different length

[0059] We screened adaptors of different lengths (28-10 mer) to identify the minimum length required to ligate efficiently the full sgRNA by splint-mediated ligation. The sequence of the two RNA fragments and the ten DNA adaptors tested are reported in Table 1.

[0060] Materials:

[0061] The following oligonucleotides were prepared by standard solid phase phosphoramidite synthesis either in house or purchased from external suppliers. The oligonucleotides prepared in house were purified by HPLC using a gradient of Acetonitrile in 0.1 M nBAAc / MeCN 95 / 5 (pH 7.5).

[0062] Table 1: Sequences of the RNA fragments and DNA splint oligonucleotides; where m = 2'-O-Methyl, * = phosphorothioate, o = phosphate

[0063] Ligation reaction

[0064] Table 2: Conditions used to test the ten different adaptors lengths

[0065] As shown in Table 2, all reactions were carried out using the same amount of RNA fragments and 12 nmol of adaptor:

[0066] - DNA adaptor 28 mer — (38-63)

[0067] - DNA adaptor 26 mer (37-62)

[0068] - DNA adaptor 24 mer (38-61)

[0069] - DNA adaptor 22 mer — (39-60)

[0070] - DNA adaptor 20 mer — (40-59)

[0071] - DNA adaptor 18 mer — (41-58)

[0072] - DNA adaptor 16 mer —>■ (42-57)

[0073] - DNA adaptor 14 mer —>■ (43-56)

[0074] - DNA adaptor 12 mer (44-55)

[0075] - DNA adaptor 10 mer — (45-54)

[0076] Where the numbers in between brackets (i.e. 37-42) indicate the starting and ending annealing point of the DNA splint on the sgRNA.

[0077] In ten different 1.5 mL vials, the RNA fragments were combined with the respective adaptor in a solution made of lx T4 RNA Ligase II reaction buffer in water / acetonitrile (89.5 / 10.5 v / v). The concentration of the RNA fragment with the 5 '-phosphate and the DNA splint were slightly in excess compared to the 5 '-end RNA fragment (the limiting reagent). On a thermomixer, the solutions were stirred at 24 °C for 10 min to allow annealing of the three oligonucleotides. T4 RNA Ligase II was then added to the solution followed by incubation at 24 °C from 2 to 24 hours.

[0078] IPC preparation

[0079] At 2 h, 40 / JL samples of the reaction mixture were taken and quenched with 2 / zL of 0.5 M EDTA (1.7 eq of EDTA compared to Mg2+). Quenching was performed on a thermomixer at 24 °C for 10 min. The solutions were then diluted to 200 / zL, filtered on an Amicon filter (500 / zL. 3 kDa cut-off, REF UFC 500396) and washed (x3 times) with deionised water to a final volume of 100 / zL. The samples were then analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry (LC-MS).

[0080] Fig. 1A shows the HPLC chromatogram of DNA adaptor 18 mer and Fig. IB shows the LC-MS chromatogram of DNA adaptor 18 mer.

[0081] Fig 2A shows the HPLC chromatogram of DNA adaptor 16 mer and Fig. 2B shows the LC-MS chromatogram of DNA adaptor 16 mer. As shown in Figure 1, the 18 mer DNA adaptor allowed full conversion of the starting RNA fragments to the full length 100 mer product. On the contrary, ligation using shorter DNA splints (below 16 nucleotides) was relatively less efficient (see Fig. 2). This was evident from the % of conversion calculated by the integrated area of the product on the HPLC spectra of the ligation crude for each adaptor, see Table 3.

[0082] Table 3: Conversion to the full length gRNA (100 mer) after 2 h expressed in % depending on the different adaptors lengths used for the ligation.

[0083] These results demonstrated that the ligation reaction is efficient when using at least an 18 mer- long splint.

[0084] Following the same IPC, the short adaptors (14 mer, 12 mer, 10 mer) were tested again with a longer incubation time. At 4 h, 40 / zL samples of the reaction mixture were taken and quenched with 2 / zL of 0.5M EDTA (1.7 eq of EDTA compared to Mg2+). Quenching was performed on a thermomixer at 24 °C for 10 min.

[0085] Table 4: Conversion to the full length gRNA (100 mer) after 4 h was expressed as % of conversion.

[0086] These results showed that by increasing the incubation time from 2 to 4 h, it is possible to achieve ligation of the full length sgRNA using short adaptors, especially the 14 mer.

[0087] Example 2: Investigation of the ligation efficiency as a function of the ligation site

[0088] The ligation efficiency was investigated as a function of the ligation site and hence of the relative length of the two sgRNA fragments to ligate. In this case, the adaptor length was kept constant and the adaptor was annealing symmetrically on the two fragments -meaning establishing 14 base pairs per segment. Table 5 shows the sequences of the RNAs fragments with their respective adaptors.

[0089] Table 5: Exemplary sequences of RNA fragments and their symmetric DNA splint oligonucleotides; where m = 2'-O-Methyl, * = phosphorothioate, o = phosphate.

[0090] Ligation reaction

[0091] Table 6 (a, b, c, d, e, f): Conditions used to test the five different combinations of RNA fragments:

[0092] Table 6(a) - TEST A

[0093] Table 6(b) - TEST B

[0094] Table 6(c) - TEST C

[0095] Table 6(d) - TEST D

[0096] Table 6(e) - TEST E

[0097] Table 6(f) - TEST CTRL

[0098] Tables 6 reports the conditions used for all the reactions tested. Each oligonucleotide used in the test had a purity ranging from 75 % to 90 %. The six test reactions were carried out in six 1.5 mL vials and the respective RNA fragments together with their adaptors mixed in solution composed of lx T4 RNA Ligase II reaction buffer in water / acetonitrile (with a volume of acetonitrile corresponding to 10.5 % of the final reaction volume). Solutions were stirred on the thermomixer at 24 °C for 10 min to allow annealing of the three oligonucleotides. T4 RNA Ligase II was then added to the solution followed by incubation at 24 °C for 24 hours.

[0099] IPC preparation

[0100] At specific time intervals (2 h, 4 h, 6 h, 24 h), 40 / zL samples of the reaction mixture were taken and quenched with 2 / zL of 0.5 M EDTA (1.7 eq of EDTA compared to Mg2). Quenching was performed on a thermomixer at 24 °C for 10 min. The solutions were then diluted to 200 / zL. filtered on an Amicon filter (500 / zL. 3 kDa cut-off, REF UFC 500396) and washed (x3 times) with deionized water to a final volume of 100 / zL. The samples were then analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry (LC-MS). In all cases (RNA 1-48 / RNA 49-100, RNA 1- 49 / RNA 50-100, RNA 1-50 / RNA 51-100, RNA 1-51 / RNA 52-100, and RNA 1-52 / RNA 53-100) the ligation product was detected although to different extents.

[0101] The efficiency of conversion was calculated from the crude HPLC trace of each test as a function of the area of the peak of the final product (the 100 mer) in [mAU*s] compared to the one of the two starting materials, see Table 7 and the related Figure 3.

[0102] Fig. 3 shows the conversion (expressed in %), over time (expressed in hours) of the different ligation tests.

[0103] Table 7: Percentage of conversion to the full length sgRNA (100 mer) according to the ligation site.

[0104]

[0105] In all the ligation tests except for test E (RNA 1-52 / RNA 53-100), the ligation reaction reached completion quickly.

[0106] Example 3: Synthesis and purification of sgRNA A and B via ligation (600 mg scale) A use test ligation reaction was performed on the 40 / / mol (600 mg) scale to identify the correct conditions required for the ligation of standard and modified sgRNA fragments in the large GMP scale. In this example two sequences — a 49mer and a 5 Imer with three 2'-O-methyl ribose phos- phorothioate modifications at the 5' and 3' end respectively — were ligated using T4 RNA ligase 2.

[0107] 3,1 Synthesis of sgRNA A

[0108] Materials:

[0109] The following oligonucleotides were prepared by standard solid phase phosphoramidite synthesis and purified by HPLC using a gradient of Acetonitrile in 0. 1 M nBAAc / MeCN 95 / 5 (pH 7.5).

[0110] Table 8. Sequences used for the ligation and final sgRNA product obtained, m = 2'-O-Methyl, r = ribonucleoside, o = Phosphate and s = phosphorothioate T4 RNA ligase 2 (product number:E00016) was purchased from Genescript. Tris HC1 (pH 7.2) reaction buffer was prepared by dissolving in 450 g of H2O: 4 g Tris HC1, 0.72 g of MgCL. 0.076 g DTT, 0. 112 g ATP, 41.3 g acetonitrile. An EDTA (pH = 8) quenching solution was prepared by dissolving in 40 mL of H2O: 9.3 g of EDTA, 2.75 mL of 28 % NaOH (w / w). 0.1 M nBAAC / MeCN (nButylaammonium acetate / MeCN 95 / 5, pH 7.5) HPLC buffer was prepared by dissolving in 4750 mL of H2O: 47 mL of nButylamine, 28.6 g of acetic acid, 250 mL of MeCN. 0.2 M Na2HPC>4, NaH2PC>4 (Phosphate buffer / MeCN, 9 / 1, pH = 6.9) desalting buffer (500 L) was prepared by adding: 437 L of WBI filtered through a Sartopore 2XLG, 10.189 kg ofNa2HPO4«2 H2O, 4.7045 kg NaH2PO4«2 H2O, 49 L of MeCN.

[0111] Methods:

[0112] The reaction for sgRNA A was set up as follow:

[0113] • 175 mL of Tris HC1 buffer,

[0114] • 0.828 g of sgRNA-69_51mer_ 3 '-fragment A in 75 ml of Tris HC1 buffer,

[0115] • 0.720 g of sgRNA-84_49mer_ 5 '-fragment A in 75 ml of Tris HC1 buffer,

[0116] • 0.464 g of sgRNA-28_28mer > splint A in 50 ml of Tris HC1 buffer,

[0117] Oligonucleotides were stirred at 25 °C at 100 rpm for 15 min. Then 1.82 mL of T4 RNA Ligase 2 (4.86 mg / mL) was added to the mixture and stirred at 25 °C at 100 rpm.

[0118] 1 mL samples were collected for IPC at 2 and 4 hours. Samples were quenched with 51 / / L of 0.5 M EDTA for 10 minutes and then diluted to 5.5 mL with H2O prior to IPC.

[0119] At the end of the ligation the solution was quenched with 18 mL of 0.5 M EDTA, stirred (100 rpm) for 15 min at 25 °C and stored at 4 °C

[0120] The solution was diafiltered on a Sartocon Hydrosart 10 kD 0. 1 m2 cassette with three liters of 0.1 M nBAAc / MeCN 95 / 5 (pH = 7.5) (internal level of the tank 300 mL). The volume was concentrated to 100 mL, the system emptied and rinsed with 100 mL of diafiltration buffer. The retentates were combined, filtered through a glass fiber filter (MN-GF5) and the filter rinsed with 100 mL of 0. 1 M nBAAc / MeCN 95 / 5 (pH = 7.5). The oligo was purified by reverse phase HPLC using the following gradient of acetonitrile in 0. 1 M nBAAc / MeCN 95 / 5 (pH = 7.5) on a YMC Triart C18-120-10 column, 20*250 mm (lot: 19863) at a flow rate of 20 mL / min, (Column oven: 50 °C, mobile phase 48 °C)

[0121] Table 9. HPLC gradient used for the purification of the ligated sgRNA

[0122]

[0123] Fractions containing the pine products were pooled providing 760 mL of Final sgRNA A with 89.43 % of purity (1028.67 mg / L content).

[0124] The solution was then concentrated to 200 mL by diafiltration on a Sartocon Hydrosart 10 kD 0.1 m2 cassette, the counterion exchanged with 2.5 L of 0.2 M Na2HPO4,NaH2PO4 (Phosphate buffer, pH = 7.0 / MeCN, 9 / 1) the concentrated solution was then diafiltrated with 2.5L Millipore Water to a permeate conductivity of 40 pS / cm and the retentate concentrated to 80 ml. The system was emptied and rinsed with 120 mL of water and the retentates combined (Final purity 89.27 %; 3689.88 mg / L). The retentate was lyophilized overnight at 0.5 mBar.

[0125] HPLC Result:

[0126] Fig. 4 shows the HPLC chromatogram of the purified sgRNA A.

[0127] Fig. 5 shows the LC-MS analysis of the purified sgRNA A.

[0128] 3,2 Synthesis of sgRNA B

[0129] Materials:

[0130] The following oligonucleotides were prepared by standard solid phase phosphoramidite synthesis and purified by HPLC using a gradient of Acetonitrile in 0. 1 M nBAAc / MeCN 95 / 5 (pH 7.5).

[0131] Table 10. Sequences used for the ligation and final sgRNA product obtained, m = 2'-O-Methyl, r = ribonucleoside, o = Phosphate and s = phosphorothioate

[0132]

[0133] T4 RNA ligase 2 (product number:E00016) was purchased from Genescript. Tris HCL (pH 7.2) reaction buffer was prepared by dissolving in 450 g of H2O: 4 g Tris HCL, 0.72 g of MgC12, 0.076 g DTT, 0. 112 g ATP, 41.3 g acetonitrile. An EDTA (pH = 8) quenching solution was prepared by dissolving in 40 mL of H2O: 9.3 g of EDTA, 2.75 mL of 28% NaOH (w / w). 0. 1 M nBAAC / MeCN (nBu- tylammonium acetate / MeCN 95 / 5, pH 7.5) HPLC buffer was prepared by dissolving in 4750 mL of H2O: 47 mL of nButylamine, 28.6 g of acetic acid, 250 mL of MeCN. 0.2 M Na2HPO4, NaH2PC>4 (Phosphate buffer / MeCN, 9 / 1, pH = 6.9) desalting buffer (500 L) was prepared by adding: 437 L of WBI filtered through a Sartopore 2XLG, 10. 189 kg of Na2HPO4«2 H2O, 4.7045 kg NaH2PO4«2 H2O, 49 L of MeCN..

[0134] Methods:

[0135] The reaction for sgRNA A was set up as follow:

[0136] • 128 mL of Tris HC1 buffer,

[0137] • 0.806 g of sgRNA-69_5 lmer_ 3 '-fragment B in 75 ml of Tris HC1 buffer,

[0138] • 0.738 g of sgRNA-91_49mer_ 5 '-fragment B in 75 ml of Tris HC1 buffer,

[0139] • 0.465 g of sgRNA-28_28mer_ splint in 75 ml of Tris HC1 buffer, Oligonucleotides were stirred at 25 °C at 100 rpm for 15 min. Then 1.83 mL of T4 RNA Ligase 2 (4.86 mg / mL) was added to the mixture and stirred at 25 °C at 100 rpm.

[0140] 1 mL samples were collected for IPC at 2 and 4 hours. Samples were quenched with 51 / / L of 0.5 M EDTA for 10 minutes and then diluted to 5.5 mL with H2O prior to IPC.

[0141] At the end of the ligation the solution was quenched with 18 mL of 0.5 M EDTA, stirred (100 rpm) for 15 min at 25 °C and stored at 4 °C.

[0142] The solution was fdtered through a glass fiber filter (MN-GF5) and the filter rinsed with 100 mL of 0. 1 M nBAAc / MeCN 95 / 5 (pH = 7.5). The solution was then diafiltered on a Sartocon Hydro- sart 10 kD 0.1 m2 cassette with three liters of 0.1 M nBAAc / MeCN 95 / 5 (pH = 7.5) (internal level of the tank 300 mL). The volume was concentrated at 100 mL, the system emptied and rinsed with 100 mL of diafiltration buffer. The retentates were combined. The oligo was purified by reverse phase HPLC using the following gradient of acetonitrile in 0.1 M nBAAc / MeCN 95 / 5 (pH = 7.5) on a YMC Triart Cl 8- 120- 10 column, 20*250 mm (lot:19863) at a llow rate of 20 mL / min, (Column oven: 50 °C, mobile phase 48 °C)

[0143] Table 11. HPLC gradient used for the purification of the ligated sgRNA

[0144] Fractions containing the pure products were pooled providing 700 mL of final sgRNA A with 85.22 % of purity (959.01 mg / L content).

[0145] The solution was then concentrated to 200 mL by diafiltration on a Sartocon Hydrosart 10 kD 0.1 m2 cassette, the counterion exchanged with 2.5 L of 0.2 M Na2HPC>4, NaH2PO4 (Phosphate buffer / MeCN, 9 / 1, pH = 7.0), the concentrated solution was then diafiltrated with 2.5L Millipore Water to a permeate conductivity of 20 uS / cm and the retentate concentrated to 80 mL. The system was emptied and rinsed with 120 mL of water and the retentates combined (Final purity 87.09 %; 3194.07 mg / L). The retentate was lyophilized overnight at 0.5 mBar.

[0146] HPLC Result:

[0147] Fig. 6 shows the HPLC chromatogram of the purified sgRNA B.

[0148] Fig. 7 shows the LCMS analysis of the purified sgRNA B.

[0149] Example 4: Multigram synthesis and purification of sgRNA A and B

[0150] Two sgRNA were prepared in the largest scale ever used for the production of sgRNA to the best of our knowledge. The sgRNA were produced on the gram scale (10-18 g) and according to GMP standards using the optimized conditions described in the use test.

[0151] 4,1 GMP production of s RNA A on gram scale

[0152] Materials:

[0153] The following oligonucleotides were prepared by standard solid phase phosphoramidite synthesis and purified by HPLC using a gradient of Acetonitrile in 0. 1 M nBAAc / MeCN 95 / 5 (pH 7.5) according to GMP standards.

[0154] Table 12. Sequences used for the ligation and final sgRNA product obtained, m = 2'-O-Methyl, r = ribonucleoside, o = Phosphate and s = phosphorothioate

[0155]

[0156] T4 RNA ligase 2 (product number:E00016) was purchased from Genescript. Tris HCL (pH 7.0) reaction buffer was prepared by dissolving in 8.6 (± 0. 1) kg of H2O: 76 (± 0.5) g Tris HCL 13.7 (± 0.1) g of MgCh, 1.5 (± 0.1) g DTT, 2.2 (± 0.1) g ATP, 780 (± 2) g acetonitrile. A 0.5 M EDTA (pH = 8) quenching solution was prepared by dissolving in 760 (± 5) g of H2O: 177 (± 1) g of EDTA, 48 (± 0.2) g of 28 % NaOH (w / v).

[0157] Methods:

[0158] The reaction for sgRNA A was set up as follows. First, the oligonucleotide solutions were prepared in IL glass bottles using the following quantities:

[0159] • 15.7 (± 0.2) g of sgRNA-69_51mer_ 3'-fragment A in 1.4 (± 0.1) kg of Tris HC1 buffer,

[0160] • 13.7 (± 0.2) g of sgRNA-91_49mer_ 5'-fragment A in 1.4 (± 0.1) kg of Tris HC1 buffer,

[0161] • 8.8 (± 0.2) g of sgRNA-28_28mer_ splint in 1.4 (± 0. 1) kg of Tris HC1 buffer,

[0162] Then, in a Downflow Booth MZ300.20.731, the solutions were added in a 13 L glass reactor at 25 °C in the following order while stirring at 45 rpm:

[0163] • 3.3 (± 0.1) kg of Tris HC1 buffer,

[0164] • Solution of sgRNA-69_5 lmer_ 3 '-fragment A

[0165] • Solution of sgRNA-9 l_49mer_ 5 '-fragment A

[0166] • Solution of sgRNA-28_28mer_ splint

[0167] The solution was stirred for 20 minutes. 35 mL (± 1) mL T4 RNA Ligase 2 (4.86 mg / mL) were added and the solution stirred at 25 °C.

[0168] 1 mL samples were collected for IPC at 2 and 4 hours. Samples were quenched with 50 / zL of 0.5 M EDTA for 10 minutes and then diluted to 5.5 mL with H2O prior to IPC.

[0169] At the end of the ligation the solution was quenched with 342 (± 2) g of 0.5 M EDTA for 20 min at 25 °C. The reaction mixture was then cooled to T = 2 °C and stirred for 60 min. The solution was then stored under nitrogen at 2 °C until diafiltration.

[0170] Purification of the product was performed using the same procedure as described in section 3.1 according to GMP standards.

[0171] 4,2 GMP production of sgRNA B on gram scale Materials: The following oligonucleotides were prepared by standard solid phase phosphoramidite synthesis and purified by HPLC using a gradient of Acetonitrile in 0. 1 M nBAAc / MeCN 95 / 5 (pH 7.5) according to GMP standards.

[0172] Table 13. Sequences used for the ligation and final sgRNA product obtained, m = 2'-O-Methyl, r = ribonucleoside, o = Phosphate and s = phosphorothioate

[0173] T4 RNA ligase 2 (product number:E00016) was purchased from Genescript. Tris HCL (pH 7.0) reaction buffer was prepared by dissolving in 8.6 (± 0. 1) kg of H2O: 76 (± 0.5) g Tris HCL, 13.7 (± 0.1) g of MgCl2, 1.5 (± 0.1) g DTT, 2.2 (± 0.1) g ATP, 780 (± 2) g acetonitrile. A 0.5 M EDTA (pH = 8) quenching solution was prepared by dissolving in 760 (± 5) g of H2O: 177 (± 1) g of EDTA, 48 (± 0.2) g of 28% NaOH (w / v).

[0174] Methods:

[0175] The reaction for sgRNA B was set up as follows. First, the oligonucleotide solutions were prepared in 2 L glass bottles using the following quantities:

[0176] • 15.3 (± 0.2) g of sgRNA-69_51mer_ 3'-fragment in 1.4 (± 0.1) kg of Tris HC1 buffer,

[0177] • 14.0 (± 0.2) g of sgRNA-91_49mer_ 5'-fragment in 1.4 (± 0.1) kg of Tris HC1 buffer,

[0178] • 8.8 (± 0.2) g of sgRNA-28_28mer_ splint in 1.4 (± 0. 1) kg of Tris HC1 buffer,

[0179] Then, in a Downflow Booth MZ300.20.731, the solutions were added in a 13 L glass reactor at 25 °C in the following order while stirring at 45 rpm:

[0180] • 2.4 (± 0.1) kg of Tris HC1 buffer,

[0181] • Solution of sgRNA-69_5 lmer_ 3 '-fragment

[0182] • Solution of sgRNA-9 l_49mer_ 5 '-fragment

[0183] • Solution of sgRNA-28_28mer_ splint

[0184] The solution was stirred for 20 minutes. 35 mL (± 1) mL T4 RNA Ligase 2 (4.86 mg / mL) were added and the solution stirred at 25 °C.

[0185] 1 mL samples were collected for IPC at 2 and 4 hours. Samples were quenched with 50 / zL of 0.5 M EDTA for 10 minutes and then diluted to 5.5 mL with H2O prior to IPC.

[0186] At the end of the ligation the solution was quenched with 350 (± 5) g of 0.5 M EDTA for 20 min at 25 °C. The reaction mixture was then cooled to T = 2-8 °C and stirred for 60 min.

[0187] Purification of the product was performed using the same procedure as described in section 3.1 according to GMP standards.

[0188] Example 5: Ligation with immobilized T4-Ligase on covalent support (Agarose CN-bromide)

[0189] Linking the enzyme on a solid support has two main advantages: i) removal of the enzyme from the reaction mixture is simple and can be achieved by filtration and ii) the enzyme can be harvested and reused for further ligation reactions To confirm if the ligation efficiency is retained once the enzyme is covalently linked onto a solid support, the ligase enzyme was first conjugated to agarose beads and the heterogeneous biocatalyst used for the sgRNA ligation reaction. Agarose beads were chosen as support of preference as their bioconjugate with the ligase enzyme provided the highest ligation efficiency compared to other supports screened (data not shown here). In particular cyanogen bromide- activated agarose beads were purchased from Sigma-Aldrich and used for the functionalization.

[0190] The primary matrix of processed agarose consists of alternating D-galactose and 3 -anhydro-galactose sugars that provide an uncharged hydrophilic matrix. For most affinity applications where harsh activation or usage conditions are needed, crosslinked agarose is favored to the non-crosslinked resin alternative. Cross-linking of the agarose does not affect the porosity significantly and increases the mechanical stability of the resin reducing enzyme leakage. In the case of cyanogen bromide-activated matrix (4% cross linked agarose), cyanogen bromide is first reacted with the hydroxyl groups on agarose to form cyanate esters and imidocarbonates (Figure 8). These groups can then be readily reacted with primary amines of ligands of interest — a ligase enzyme in the case of this patent — under very mild conditions. This reaction can generate either: i) a stable and uncharged imidocarbonate linkage or ii) a less stable and charged isourea bond where a small but constant leakage can occur. In general CNBr-matrixes have several advantages including mild functionalization pH and a relatively simple and reproducible coupling procedure.

[0191] Fig. 8 shows the graphical representation of the functionalization of a cyanogen bromide-activated matrix.

[0192] 5,1 Immobilization on the Agarose CN -bromide

[0193] To couple the enzyme with the resin, four buffers need to be prepared first: i) the coupling buffer, used to dissolve the protein for the coupling; ii) the activated buffer, required to wash and swell the resin while preserving the activity of the reactive groups (cyanate ester or imidocarbonate), which will otherwise hydrolyze at high pH. The manufacturer resin contains lactose which is necessary to stabilize the beads for the lyophilization process; however, lactose will interfere with the binding if present during coupling and need to be removed; iii) the blocking buffer to block unreacted groups after the coupling and finally iv) the washing buffer to remove by extensive wash the blocking solution. Table 14 shows in detail the preparation of the four buffers.

[0194] Table 14: Detailed preparation of the four buffers of interest. Coupling buffer:

[0195] Activated buffer: Blocking buffer:

[0196] Washing buffer:

[0197] For the coupling reaction, 2.5 g of beads were weighted in a 50 mL falcon tube. The resin was then washed and swelled on a shaker in c.a. 200 mL of cold activated buffer (ImM HC1) for 30 minutes. The resin was then fdtered and washed on a sintered glass fdter (porous size G3) using c.a. 80 mL of distilled water.

[0198] In the meantime, the T4 RNA-ligase 2 was dissolved in the coupling buffer (0. IM NaHCO3, 0.5M NaCl pH 8.4) to a concentration of 4.86 mg / ml. 10 mL of T4 RNA-ligase 2 (48.6 mg) was coupled to the washed resin on a shaker for 2 h at room temperature. The ligand excess was washed away using the coupling buffer. Any remaining active groups were blocked using a blocking / quenching buffer (1 M ethanolamine pH 8) for 2 h at room temperature. The resin was then washed thoroughly three times alternating one wash with the coupling buffer (pH 8) to one wash with the washing buffer (pH 4).

[0199] Finally, the product was spirited in two falcon tubes (c.a. 25 mL each). One tube was stored at 4 °C in aqueous buffer to be used subsequently for example 5.2. The other tube was instead lyophilised and used for example 5.3 in order to understand the different ligation efficiency upon the two different storage conditions for the enzyme.

[0200] 5,2 Ligation using the heterogenous T4-Ligase catalyst stored in SOLUTION

[0201] The ligation was tested using the immobilized enzyme either stored in solution or as lyophilized solid in order to understand: i) if the ligation efficiency is conserved when the enzyme is covalently linked onto the Agarose CN-bromide support; and ii) the best way of storing the enzyme. Table 15 shows in detail the conditions for the ligation using the immobilized enzyme in solution. Table 15: Conditions for the ligation using the immobilized enzyme in solution.

[0202] In a 1.5 mL vial, the RNA fragments and the DNA adaptor were combined in a solution made of 200 / zL T4 RNA Ligase II reaction buffer and 1151.8 pL water (total reaction volume of 2000.0 / zL). The solution was stirred at 24 °C on the thermomixer for 10 mins. 320 / zL of immobilized T4 RNA Ligase II on agarose beads stored in solution was then added and the reaction incubated at 24 °C for 24 hours.

[0203] IPC preparation

[0204] At specific time intervals (2h, 4h, 6h, 24h), 40 pL samples of the reaction mixture were taken and quenched with 2 pL of 0.5M EDTA (1.7 eq of EDTA compared to Mg2+). Quenching was performed on a thermomixer at 24 °C for 10 min. The solutions were then diluted to 200 pL, filtered on an Amicon filter (500 pL, 3 kDa cut-off, REF UFC 500396) and washed (x3 times) with deionised water to a final volume of 100 pL.

[0205] 5,3 Ligation using the heterogenous T4-Ligase catalyst stored as LYOPHILISED solid

[0206] The ligation efficiency was tested using the immobilized enzyme stored as lyophilised solid. Table 16 shows in detail the conditions used for the ligation with the immobilized enzyme stored as lyophilised solid.

[0207] Table 16: Conditions used for the ligation using the immobilized enzyme in lyophilized form.

[0208] In a 1.5 mL vial, the RNA fragments and the symmetric adaptors were combined in a solution made of 200 / zL T4 RNA Ligase II reaction buffer together and 1446.8 / zL water for a total reaction volume of 2000.0 / zL. The solution was stirred at 24 °C on a thermomixer for 10 mins. 25 mg of T4 RNA Ligase II immobilized on agarose beads stored as lyophilized solid was weighted and added to the solution followed by incubation at 24 °C for 24 hours.

[0209] IPC preparation

[0210] At specific time intervals (2 h, 4 h, 6 h, 24 h), 40 / zL samples of the reaction mixture were taken and quenched with 2 / zL of 0.5M EDTA (1.7 eq of EDTA compared to Mg2+). Quenching was performed on a thermomixer at 24 °C for 10 min. The solutions were then diluted to 200 / zL. filtered on an Amicon filter (500 / zL. 3 kDa cut-off, REF UFC 500396) and washed (x3 times) with deionised water to a final volume of 100 / zL.

[0211] 5.4 Reusing the immobilized enzymes (same batch) multiple times over 4 days

[0212] We evaluated the possibility of reusing the immobilized enzyme multiple times after a ligation reaction and we analyzed the retention of the enzyme activity upon the steps required to isolate, store and resuspend the enzyme. This analysis was performed both for the heterogenous biocatalyst stored in solution or stored as lyophilized solid. The procedures for the isolation and storage of the two batches were as follows. In the case of the enzyme stored in solution, the ligation mixture was first centrifuged and the supernatant containing the ligation product, buffer, and any trace of starting material removed. The solid — i.e. the immobilized enzyme — was then washed once with 2 mL of water, centrifuged again and finally stored in solution at 4 °C at the same concentration used previously for storage (2.5 mg / mL, cf Example 5.2).

[0213] In the case of the enzyme stored as lyophilized powder the same washing steps were performed (centrifugation of the ligation solution, washing with 2 mL and centrifugation), but the solid was instead lyophilized overnight prior to being used the day after. The ligation reactions were then repeated every day for 4 days using the same conditions outlined in examples 5.2 and 5.3 with the enzymes stored in the respective conditions — solution or lyophilized. Samples of the ligation reactions were collected at 2 h, 4 h, and 6 h and quenched using the same procedure previously described (cf. IPC preparation example 5.2 and 5.3).

[0214] The enzyme activity was retained also after 4 days of storage as visible from the % of the area of the ligation product [mAU*s] compared to the starting materials, see Table 17 and Figure 9. The conversion was calculated from the HPLC analysis of each time sample.

[0215] Table 17: Conversion to the full length gRNA (100 mer) expressed in % at 2 h, 4 h, and 6 h time points for each ligation performed over 4 days using either the enzyme stored in solution or as lyophilized solid.

[0216]

[0217] Fig. 9 shows the conversion (expressed in %), over time (expressed in hours) of the two differently stored enzymes linked to agarose beads.

[0218] Fig. 10 shows the LC-MS chromatograms of the reaction crude from a 2h ligation using the immobilized enzyme stored in solution respectively on the 1st day (Fig. 10A) and 4th day (Fig. 10B).

[0219] Fig. 11 shows the LC-MS chromatograms of the reaction crude from a 2h ligation using the immobilized enzyme stored as lyophilized solid respectively on the 1st day (Fig. HA) and 4th day (Fig. 11B).

[0220] These results demonstrated that the ligation efficiency decreased over multiple isolations and reuse of the same enzyme batch (Figure 10 and 11). The ligation reaction is faster with the enzyme kept in solution compared to the lyophilized one.

[0221] Example 6: Investigation of the ligation with different quantities of enzyme

[0222] Materials:

[0223] The following oligonucleotides were prepared by standard solid phase phosphoramidite synthesis and purified by HPLC using a gradient of Acetonitrile in 0. 1 M nBAAc / MeCN 95 / 5 (pH 7.5) according to GMP standards.

[0224] 6,1 Enzyme quantity optimization

[0225] The effect of enzyme quantity on ligation efficiency was analyzed on a small scale. Different amounts of enzyme were used to ligate two 20-mer sequences corresponding to the 20 bases at the left and right of the ligation point for the sgRNA.

[0226] Table 18: Conditions used for screening the effect induced by enzyme quantity on ligation efficiency.

[0227]

[0228] For the purpose of the test, different amounts of enzymes were screened n = 10, 25, 50, 75, 100, 125, 170 U. In all cases the oligonucleotides were hybridized at 23 °C for 5 min prior to the addition of the buffer and the enzyme. The reactions were left stirring into a thermoshaker for 1.5 h at room temperature and then analyzed by LC-MS. In the case of the reagents not being depleted the reaction was left for an additional amount of time (see below).

[0229] When more than 75 U of enzymes were used for the reaction, the ligation proceeded to completion after 1.5 h. Reactions with lower enzyme units (10, 25, 50 U), however, required longer incubation times to go to completion causing the formation of other impurities.

[0230] 6,2, Ligation stress test at different amounts of enzyme

[0231] A stress test was performed varying the quantity of enzyme added (± 10% and ± 50%) to understand if errors or variations in the amount of enzyme could affect the ligation efficiency.

[0232] 6,2, 1 Ligation efficiency according to a ± 10% variation in enzyme quantity

[0233] Ligations reactions were set up into a final volume of 5.090 mL according to the following table to understand the effect in a ± 10% variation on enzyme quantities:

[0234] Table 19. Conditions used to screen the effect of ± 10% variation of enzyme.

[0235] As described in the previous examples, the mixture of the three oligonucleotides was stirred in the buffer at 100 rpm for ca. 10 min at room temperature to allow annealing of the three strands. The enzyme was then added according to the quantity specified in the figure 12 and left stirring for 3.5 hours at 25 °C. The reaction was monitored after 2 h and 3.5 h. No significant variation (>2%) from standard conditions observed even in the presence of -10% less of T4 RNA Ligase 2.

[0236] Fig. 12 shows the variation in the conversion to products when the enzyme loading is increased or decreased of 10%.

[0237] 6,2,2 Ligation efficiency according to a ± 50% variation in enzyme quantity

[0238] Ligations reactions were set up into a final volume of 5.090 mL according to the following table to understand the effect of a ±50% variation on enzyme quantity:

[0239] Table 20: Conditions used to screen the effect of ±50% variation of enzyme amount.

[0240] As described in the previous examples, the mixture of the three oligonucleotides was stirred in the buffer at 100 rpm for ca. 10 min at room temperature to allow annealing of the three strands. The enzyme was then added according to the quantity specified in the figure 13 and left stirring for 4 hours at 25 °C. The reactions were monitored after 2 h, 4 h and 7 h (the latter was monitored only in the case of -50% of enzyme). No significant variation (>2%) in the ligation efficiency was observed in the presence of +50% of T4 RNA Ligase 2, while the reaction with -50% of T4 RNA Ligase 2 proceeded slightly slower (4% less of product at 4h) but still yielding the full size product.

[0241] Fig. 13 shows the variation in the conversion to products when the enzyme loading is increased or decreased of 50%.

[0242] Example 7: Effect of sgRNA fragment and splint concentrations on ligation

[0243] Materials:

[0244] The following oligonucleotides were prepared by standard solid phase phosphoramidite synthesis and purified by HPLC using a gradient of Acetonitrile in 0. 1 M nBAAc / MeCN 95 / 5 (pH 7.5) according to GMP standards.

[0245] 7,1 Ligation efficiency and its dependency on different equivalents of sgRNA fragment.

[0246] For this example, the ligation was tested using two 20-mer fragments corresponding to 20 bases at either side of the ligation point chosen on the sgRNA. The ligation was screened at different equivalents of the sgRNA fragments or of the splint. The following four reaction conditions were screened where: i) the 3'-sgRNA fragment to ligate is the limiting reagent; ii) the 5'-sgRNA fragment to ligate is the limiting reagent; iii) the two sgRNA fragments and the splint are in equimolar amounts; iv) the 3'- end fragment and the splint are only slightly in excess compared to the 5 '-end fragment.

[0247] Reactions were set up in a total volume of 100 / zL using the quantities outlined in the following table.

[0248] The mixture of the three oligonucleotides was left to hybridize for 5 min at room temperature prior to addition of the enzyme. The enzyme was then added according to the quantity specified in the table and left stirring for 1.5 hours at 23 °C. All ligation reactions went to completion after 1.5 hours of incubation.

[0249] 7,2 Ligation efficiency and its dependency on different concentrations of sgRNA fragments.

[0250] For this example, the ligation was tested using the two fragments which form the full size sgRNA upon ligation. The ligation was screened at two different concentrations of the sgRNA fragments.

[0251] Reactions were set up in a total volume of 100 / zL using the quantities outlined in the following table.

[0252]

[0253] The mixture of the three oligonucleotides was left to hybridize for 5 min at room temperature prior to addition of the buffer and the enzyme. The buffer and the enzyme were then added according to the quantity specified in the table and left stirring for 2 hours at 23 °C. Both ligation reactions went to completion after 1.5 hours of incubation.

[0254] Example 8: Buffer composition

[0255] The effect of the buffer composition on ligation efficiency was analyzed. Different tests were performed changing the concentration of one or more parameters in the buffer. In particular, the concentration of the following buffer components was screened: i) bivalent metal ion; ii) ATP; iii) acetonitrile.

[0256] The primary buffer salts were outsourced externally. Dithiothreitol (DTT) (APOBIMB1015- 100g) was purchased from Apollo; Magnesium chloride (M4880-100G) from Sigma- Aldrich Chemie GMBH; ATP (Adenosine 5 '-triphosphate disodium salt) (UBP-P1040-100-100G) ftom Lucema-Chem AG; and finally, Tris HCL (hydroxymethyl)aminomethane hydrochloride, (CAS: 1185-53-1) (ACR0228030010) ftom Acros organics.

[0257] The conditions used to screen the effect induced by different salt concentration on ligation efficiency are shown in Table 21.

[0258] Table 21 : Conditions used to screen the effect induced by different salt concentration on ligation efficiency.

[0259] As control, the standard lOx T4 RNA Ligase 2 buffer, purchased from NEB has been used. Its composition is as follow:

[0260] In all cases, for each test the oligonucleotides were hybridized at 23 °C for 5 min prior to the addition of the buffer and the enzyme. Concentration of the buffer in the final ligation reaction is lx. The reactions were left stirring into a thermoshaker for 1.5 h and 8 h at room temperature and then analyzed by LC-MS.

[0261] 8, 1 Bivalent metal ion concentration

[0262] For the purpose of the test, different concentrations of MgCh were screened n = 5, 10, 15 rnM (final concentration in the lx ligation buffer). The concentrations of the other salts were kept constant according to the standard T4 RNA Ligase 2 buffer, respectively Tris HCL 50 mM; DTT 1 mM; ATP 0.4 mM; pH of 7.5 (final concentration in the lx ligation buffer). All the lOx buffers were prepared in house.

[0263] The reactions were monitored after 1.5 h, and 8 h. The ligation efficiency works better when using the buffer with a salt concentration of 15 mM bivalent metal ion. The product yield is higher compared to the other buffer compositions screened. 15 mM of MgCL2is considered the best condition.

[0264] 8,2 ATP concentration

[0265] In parallel to the different bivalent metal ion concentrations (see paragraph 8. 1), different concentrations of ATP were screened n = 0.75, 1, 1.25 mM (final concentration in the lx ligation buffer). The concentrations of the other salts were kept constant according to the standard T4 RNA Ligase 2 buffer, respectively Tris HCL 50 mM; MgCL22 mM; DTT 1 mM; pH of 7.5 (final concentration in the lx ligation buffer). The lOx buffers were prepared in house.

[0266] As described in the previous example, the reactions were monitored after 1.5 h, and 8 h. No significant variation in ligation efficiency was observed by increasing the ATP concentration, which seems to not play a role in the ligation reaction to the same extent as the bivalent metal ion concentration.

[0267] 8,3 ATP concentration at fixed metal ion

[0268] Keeping the concentration of the metal ion (MgCL215 mM in lx buffer) constant, different concentrations of ATP were screened; being aware that the bivalent metal ion concentration is now higher than the one of the standard T4 RNA Ligase 2 buffer (from NEB) (see paragraph 8.2). Again, the screened ATP concentrations were n = 0.75, 1, 1.25 mM (final concentration in the lx ligation buffer). With a pH of 7.5, the lOx buffers were prepared in house.

[0269] As described in the previous examples, the reactions were monitored after 1.5 h, and 8 h. No significant variation in the ligation efficiency was observed by increasing the ATP concentration at fixed (higher) divalent metal ion. Indeed, ATP seems to not play any role, independently of the MgC12 concentration.

[0270] With a pH of 7.5, the lOx ligation buffer for the all ligation test in small scale (see examples before) was prepared in house. The final buffer salts concentrations were set as shown in the tables below.

[0271] 8,4 Acetonitrile (ACN) concentration

[0272] For the purpose of the test, different percentages of ACN were screened n = 10.5%, 12.5%, 15%.

[0273] The concentrations of the other buffer salts were determined according to the optimized “in house” T4 RNA Ligase 2 buffer (see example 8.3), respectively Tris HCL 500 mM; MgC12 150 mM; DTT 10 mM; ATP 4 mM. With a pH of 7.5, the lOx buffers were prepared in house.

[0274] For this experiment, T4 RNA ligase 2 (product number: U747GHC090-2) was purchased from Genscript. The different conditions used for screening the effect induced by the ACN concentration of the buffer composition on ligation efficiency are shown in Table 22.

[0275] Table 22: Conditions used for screening the effect induced by ACN concentration of the buffer composition on ligation efficiency.

[0276] The mixture of the three oligonucleotides was stirred in the buffer at 100 rpm for ca. 10 min at room temperature to allow annealing of the three strands. The enzyme was then added and left stirring on Easymax for 4 hours at 25 °C. The reactions were monitored after 2 h, 4 h and 6 h (the latter was monitored only in the case of 15% ACN - test C) (see figure 14)

[0277] Increasing the percentage of ACN up to 15% in the reaction, a significant variation of the final product conversion — with a decrease of 40% from standard conditions — was observed. Likely ACN inhibits the activity of the enzyme.

[0278] Fig. 14 shows the variation in the conversion to products when the acetonitrile content is increased up to 15%.

[0279] Example 9: Buffer optimization via additives

[0280] Different additives can be added to the standard buffer to increase ligation efficiency. The following additives were tested at different concentrations and the ligation efficiency reported as % of conversion: i) bivalent metal ion: Mn2+; ii) DMSO; iii) PEG8000.

[0281] The conditions used to screen the effect induced by different salt concentration on ligation efficiency are shown in Table 23. Table 23: Conditions used to screen the effect induced by different salt concentration on ligation efficiency.

[0282] As control, the following optimized lOx T4 RNA Ligase 2 buffer (buffer was prepared in house, see Example 8. 1) was used:

[0283] In the experiments, the oligonucleotides were hybridized at 23 °C for 5 min prior to addition of the buffer and the enzyme. The reactions were then left stirring into a thermoshaker at room temperature for 15, 30, 60, 90, 120, 240 min and then analyzed by LC-MS.

[0284] 9, 1 Bivalent metal ion concentration

[0285] For the purpose of the test, different concentrations of MnCL (used in place of MgCL) were screened: [C] = 1, 2, 5, 10, 15 mM (final concentration in the lx ligation buffer). The concentration of the other salts were kept constant and identical to the standard lx T4 RNA Ligase 2 buffer: Tris HCL 50 mM; DTT 1 mM; ATP 0.4 mM; pH of 7.5. All lOx buffers were prepared in house.

[0286] Reactions were monitored after 15, 30, 60, 90, 120, 240 min.

[0287] Beyond 5 mM MnC12, ligation efficiency and conversion to the final product drops down. Ligation efficiency is relatively increased instead at low concentration of the bivalent metal ion (1 mM). In this case, % of conversion to the final product is higher compared to the other buffer compositions.

[0288] 9,2 DMSO percentage

[0289] In parallel to the bivalent metal ion (see paragraph 9.1), different percentages (v / v) of DMSO as additive to the buffer were screened n = 1 %, 5 %, 10 %. Concentrations of the other salts were kept constant and identical to the optimized lx T4 RNA Ligase 2 buffer: Tris HCL 50 mM; MgCL 15 mM; DTT 1 mM; ATP 0.4 mM; pH of 7.5. The lOx buffers were prepared in house.

[0290] Reactions were monitored after 2 h, and 4 h. No significant variation in ligation efficiency was observed across the different buffers indicating that DMSO does not play a pivotal role in the ligation reaction.

[0291] 9,3 PEG percentage

[0292] Different percentages (n = 5 %, 15 %, w / v) of PEG8000 were also screened to understand the effect of crowding agents on ligation efficiency. Concentrations of the other salts were kept constant and identical to the standard lx T4 RNA Ligase 2 buffer: Tris HCL 50 mM; MgCf 15 mM; DTT 1 mM; ATP 0.4 mM; pH of 7.5. The buffers were prepared in house.

[0293] Reactions were monitored after 2 h, and 4 h. No significant variation in ligation efficiency was observed by increasing the PEG8000 percentage indicating that PEG8000 does not play a major role in the ligation reaction.

Claims

Claims1. A method of synthesizing a single guide RNA (sgRNA) comprising: providing a first single stranded RNA fragment comprising a terminal region with a 3 '-hydroxyl group and a second single stranded RNA fragment comprising a terminal region with a 5' phosphate moiety, wherein the first and second RNA fragments have a length of 45 - 51 nucleotides (nt), providing a single stranded DNA oligonucleotide having a length from 18 - 28 nt, wherein the DNA oligonucleotide comprises a first region being complementary to the first RNA fragment at its 3 '-hydroxyl group end and a second region being complementary to the second RNA fragment at its 5 '-phosphate moiety end, wherein each of the two complementary regions of the DNA oligonucleotide have at least a length of 9 nucleotides.Hybridizing the two RNA fragments and the DNA oligonucleotide to form a complex and ligating the first and the second RNA fragment at a ligation site present between the two RNA fragments using a ligase to synthesize the sgRNA.

2. The method of claim 1, wherein the sgRNA has a length of 100 nt.

3. The method of claim 1 or 2, wherein the first RNA fragment has a length of 49 nt and the second RNA fragment has a length of 51 nt or the first RNA fragment has a length of 51 nucleotides and the second RNA fragment has a length of 49 nt.

4. The method of claims 1 - 3, wherein the two complementary regions of the DNA oligonucleotide have the same length.

5. The method of claims 1 - 3, wherein the DNA oligonucleotide has a length of 26 nt and each of the two complementary regions has a length of 13 nt.

6. The method of claims 1 - 3, wherein the DNA oligonucleotide has a length of 28 nt and each of the two complementary regions has a length of 14 nt.

7. The method of claims 1 - 4, wherein the two complementary regions of the DNA oligonucleotide are 100% complementary to the respective RNA fragment regions.

8. The method of claims 1 - 5, wherein the sgRNA comprises a crRNA segment and tra- crRNA segment.

9. The method of claim 8, wherein the ligation site lies within the tracrRNA segment.

10. A reaction mixture for the large-scale synthesis of sgRNA comprising:0.05 - 0.2 rnM single stranded oligonucleotides as defined in claims 1 -8,0.05 - 0.2 rnM single stranded DNA oligonucleotide as defined in claims 1 - 8,0.001 mg / ml - 0.5 mg / ml ligase, in an aqueous solution at about pH 7.0, wherein the reaction mixture has a volume of at least 10'5liters to 10000 liters.

11. The reaction mixture of claim 10, wherein the volume is at least 1 to 5000 liters, preferably 5 to 2500 liters, more preferably 10 to 1000 liters.

12. The reaction mixture of claim 10 or 11 further comprising the additives acetonitrile, ATP, DTT, PEG, manganese or magnesium and DMSO.

13. The reaction mixture of claims 10 - 12, wherein the ligase is a RNA ligase, preferably a T4 RNA ligase 2.

14. The reaction mixture of claims 10 - 13, wherein the enzyme was immobilized on agarose beads to ease separation of the ligation products and reuse of the enzyme for subsequent reactions.

15. The reaction mixture of claims 10 - 14, comprising:I O g to 50g single stranded oligonucleotides,I O g to 50g single stranded DNA oligonucleotidesI g to 200mg ligase in a volume of 20 / d - 7 liters.

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