Grna and biosynthesis method therefor

Abstract

Provided are a gRNA and a biosynthesis method therefor, suitable for the field of gRNA synthesis. The biosynthesis method comprises: connecting the 3' end and the 5' end of different substrates by using an RNA ligase to form a phosphodiester bond, so as to form a gRNA. The gRNA comprises natural gRNA or non-natural gRNA. The number of the substrates is greater than or equal to 2. The connection site of the substrate is located at any one or more positions in the stem-loop structure complementary region, the stem-loop structure annular region or a linker region between the stem-loop structures of the gRNA, and base complementary pairing can be carried out in a substrate or among substrates to form a secondary structure of the gRNA; wherein the RNA ligase comprises any one or more enzymes in the RNA ligase family 1, 2 or 3. The solution solves the problem in the prior art of low purity of synthesized gRNA.

Description

gRNA and biosynthesis method thereof

[0001] This application is based on the Chinese application with CN application number 202311335106.2 and application date October 16, 2023, and claims its priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of gRNA synthesis, and in particular to a gRNA and a biosynthesis method thereof. Background Art

[0003] Guide RNA (gRNA) is a non-coding RNA discovered in the mitochondria of the kinetoplastid trypanosome in the 1980s. It serves as a guide in the RNA editing process. gRNA has gained widespread attention since the discovery and application of CRISPR-Cas9 gene editing technology. This technology uses sgRNA (single guide RNA) to precisely locate the desired editing site on DNA—the PAM (protospacer adjunct motif) site. Cas9 (DNA endonuclease) then locates the corresponding site and cuts it, achieving the desired gene editing (deletion, insertion, or modification). The gRNA is a single-stranded RNA strand of approximately 67-142 nt long, consisting of both crRNA and tracrRNA. The crRNA is a variable sequence of approximately 15-25 nt that specifically recognizes the target gene through complementary pairing. The tracrRNA is a fixed sequence responsible for recruiting Cas9, and its sequence varies depending on the type of Cas9.

[0004] Currently, the most commonly used Cas9 protein is SpCas9, derived from Streptococcus pyogenes. Other available Cas9 proteins include SaCas9 from Staphylococcus aureus, FnCas9 from Francisella novicida, CjCas9 from Campylobacter jejuni, CdCas9 from Corynebacterium diphtheriae, St1Cas9 from Streptococcus thermophilus, and NmeCas9 from Neisseria meningitidis. The commonly used gRNAs for SpCas9, SaCas9, FnCas9, and CjCas9 are approximately 100 nt in length, while those for St1Cas9 and CdCas9 are approximately 115 nt in length. The longest gRNA available for NmeCas9 is approximately 140 nt. Although the gRNA lengths required for each Cas9 protein vary, all gRNAs have similar structures.

[0005] For gRNAs with a chain length of about 100 nt, they can be synthesized through in vitro transcription and solid-phase synthesis in the existing technology. However, the existing solid-phase synthesis method is mainly achieved through primer synthesis, and the synthesis scale is limited to the nmol-μmol level, which is difficult to scale up to production scale. And due to the characteristics of solid-phase synthesis, the increase in chain length will lead to a decrease in purity and yield, and there will be many problems such as reduced coupling efficiency, decreased physical stability of the carrier, and increased side chain impurities. For gRNA prepared by in vitro transcription, the terminal sequence of the gRNA product is inaccurate, affecting product quality.

[0006] Summary of the Invention

[0007] The main purpose of the present invention is to provide a gRNA and a biosynthesis method thereof to solve the problem of low purity of synthesized gRNA in the prior art.

[0008] To achieve the above-mentioned object, according to a first aspect of the present invention, a method for the biosynthesis of gRNA is provided, which comprises: using RNA ligase to connect the 3′ end and the 5′ end of different substrates to form a phosphodiester bond to form a gRNA, wherein the gRNA comprises a natural gRNA or a non-natural gRNA, and the number of substrates is ≥2; the connection site of the substrate is located at any one or more positions in the complementary region of the stem-loop structure, the ring region of the stem-loop structure, or the connecting region (linker) between the stem-loop structures of the gRNA, and base complementary pairing can be performed inside the substrate or between the substrates to form the secondary structure of the gRNA; the RNA ligase comprises any one or more enzymes of RNA ligase family 1, 2 or 3.

[0009] Furthermore, the RNA ligase includes one or more of the RNA ligases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24; or The RNA ligase shown in any one of NO: 24 has an enzyme with 70% or more identity, preferably 70% or more identity, 75% or more identity, 80% or more identity, 90% or more identity, 95% or more identity, 99% or more identity, 99.5% or more identity, or 99.9% or more identity.

[0010] Furthermore, the substrate includes 2 or more.

[0011] Furthermore, the length of the gRNA is 67-146 nt.

[0012] Furthermore, the length of the gRNA is 91-104 or 130-146 nt.

[0013] Furthermore, the sequence of the gRNA includes the nucleotide sequence shown in any one of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 43.

[0014] Further, the substrate includes SEQ ID NO: 29 or a nucleotide sequence consisting of 15-25 N bases; and the nucleotide sequences shown in SEQ ID NO: 30 and SEQ ID NO: 31; or a sequence consisting of a nucleotide sequence consisting of 15-25 N bases and a nucleotide sequence shown in SEQ ID NO: 36, or a nucleotide sequence shown in SEQ ID NO: 32, and a nucleotide sequence shown in SEQ ID NO: 33; a nucleotide sequence consisting of 15-25 N bases located 5' to SEQ ID NO: 36; or a sequence consisting of a nucleotide sequence consisting of 15-25 N bases and a nucleotide sequence shown in SEQ ID NO: 47, or a nucleotide sequence shown in SEQ ID NO: 44, and a nucleotide sequence shown in SEQ ID NO: 45 and SEQ ID NO: 46; a nucleotide sequence consisting of 15-25 N bases located 5' to SEQ ID NO: 36; or a sequence consisting of a nucleotide sequence consisting of 15-25 N bases and a nucleotide sequence shown in SEQ ID NO: 50, or a nucleotide sequence shown in SEQ ID NO: 48, and a nucleotide sequence shown in SEQ ID NO: The nucleotide sequence shown in NO: 49; the nucleotide sequence consisting of 15-25 N bases is located in the 5' direction of SEQ ID NO: 50.

[0015] Furthermore, when there are two substrates, the biosynthesis method includes: mixing the substrates and RNA ligase, where the substrates include a first substrate and a second substrate, and the RNA ligase connects the phosphate group at the 5' end of the first substrate and the hydroxyl group at the 3' end of the second substrate to form a phosphodiester bond to obtain gRNA.

[0016] Furthermore, the 5' end of the second substrate is a 5' protecting group, and the 3' end of the first substrate is a 3' protecting group.

[0017] Furthermore, when there are three substrates, the substrates include a first substrate, a second substrate and a third substrate, and the biosynthesis method includes: mixing the first substrate, the second substrate and RNA ligase, the 5' end of the first substrate is a phosphate group, the 3' end of the first substrate is a 3' protecting group, the 5' end and 3' end of the second substrate are hydroxyl groups, the RNA ligase connects the phosphate group at the 5' end of the first substrate and the hydroxyl group at the 3' end of the second substrate to form a phosphodiester bond, and obtains an intermediate product with a hydroxyl group at the 5' end; 5' phosphorylation is performed on the intermediate product to obtain a 5' phosphorylated intermediate product; the 5' phosphorylated intermediate product, the third substrate and RNA ligase are mixed, and the phosphate group at the 5' end of the 5' phosphorylated intermediate product and the hydroxyl group at the 3' end of the third substrate are connected to form a phosphodiester bond to obtain gRNA; wherein, the 5' end of the third substrate is a 5' protecting group.

[0018] Furthermore, the biosynthesis method is repeatedly used to connect and 5' phosphorylate four or more substrates multiple times, with two substrates connected each time, and the gRNA is obtained by connecting them in sequence.

[0019] Furthermore, the template nucleic acid chain is used to guide the connection of the substrate; the template nucleic acid chain has at least 3 base-specific binding to different substrates, and the bases of the substrate specifically bound to the template nucleic acid chain are adjacent and form a nick, thereby obtaining a double-stranded nucleic acid structure containing the nick, and the adjacent bases at both ends of the nick are the 5' end and 3' end of different substrates, respectively, the 5' end is a 5' phosphate group, and the 3' end is a hydroxyl group; RNA ligase is used to connect the 5' phosphate and hydroxyl groups upstream and downstream of the nick to form a phosphodiester bond to obtain a double-stranded nucleic acid structure; DNA enzyme is used to digest the template nucleic acid chain on the double-stranded nucleic acid structure to obtain gRNA.

[0020] Furthermore, the sequence of the template nucleic acid chain includes any one of the nucleotide sequences shown in SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID NO: 51.

[0021] Furthermore, after the template nucleic acid chain is mixed with different substrates, it is incubated at high temperature and then slowly cooled and annealed or incubated at a constant temperature to obtain a double-stranded nucleic acid structure containing a nick; the temperature of the constant temperature incubation includes 4 to 37°C, and the constant temperature incubation time is ≥10 minutes; the slow cooling and annealing after high temperature incubation includes: incubation at 95°C for 2 minutes, cooling to 12°C at 0.5 to 1.2°C / min, incubation at 12°C for 10 minutes and then cooling to 4°C.

[0022] Furthermore, the 3′ end of the substrate located at the 3′ end of the gRNA is a 3′ protecting group, and the 5′ end of the substrate located at the 5′ end of the gRNA is a 5′ protecting group.

[0023] Furthermore, the secondary structure of gRNA is formed by spontaneous base pairing between substrates or between substrates.

[0024] Furthermore, 3′ protecting groups include but are not limited to N-acetylgalactosamine group, phosphate group, -O-NH2, azido group, cyanoethyl group, allyl group or 2-nitrobenzyl group and other modification groups that can prevent the formation of phosphate bond; 5′ protecting groups include but are not limited to hydroxyl group, hydrogen group or methyl group and other modification groups that can prevent the formation of phosphate bond.

[0025] Furthermore, the substrate includes one or more non-natural nucleotides, and the non-natural nucleotides include: ribonucleotides having one or more of ribose 2' position modification, ribose backbone modification, base modification or phosphate backbone modification; ribose 2' position modification includes 2'-methoxy modification, 2'-fluorine modification, 2'-hydrogen modification, 2'-methoxyethyl modification, 2'-FANA modification, locked nucleic acid modification or hexitol nucleic acid modification; ribose backbone modification includes replacing the ribose in the nucleotide with ribuloNA, TNA, tPhoNA, or dXNA; base modification includes deazaadenine C7 modification, deazaguanosine C7 modification, cytosine C5 modification or uridine C5 modification; phosphate backbone modification includes PS modification.

[0026] In order to achieve the above-mentioned object, according to the second aspect of the present invention, a gRNA is provided, which includes the gRNA prepared by the above-mentioned biosynthesis method.

[0027] By applying the technical solution of the present invention and utilizing the above-mentioned biosynthetic method, two or more substrates are connected by RNA ligases of the Rnl1, Rnl2, or Rnl3 families. The nicks for connection can be located on the stem-loop structure of the gRNA or in the connecting region between the stem-loop structures, thereby connecting multiple substrates into a complete RNA chain, and the substrates can spontaneously perform base complementary pairing, thereby preparing a gRNA with a secondary structure. Utilizing the above-mentioned biosynthetic method, the reaction can be carried out at a higher substrate concentration, resulting in a higher yield, which can meet the requirements of industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0029] Figure 1 shows a schematic diagram of the reactions according to Examples 1-3 of the present invention; wherein A is a schematic diagram of splicing three fragments using RNA ligase, B is a schematic diagram of splicing two fragments using RNA ligase; and C is a schematic diagram of splicing fragments using RNA ligase under the guidance of a template DNA chain.

[0030] FIG2 shows a schematic diagram of the secondary structure of SEQ ID NO: 26 according to Example 1 of the present invention.

[0031] FIG3 shows a schematic diagram of the secondary structure of SEQ ID NO: 28 according to Example 1 of the present invention.

[0032] FIG4 shows a graph showing the gel electrophoresis results of step 1 according to Example 1 of the present invention.

[0033] FIG5 shows a UPLC result diagram of step 1 according to Example 1 of the present invention.

[0034] FIG6 shows a UPLC result diagram of step 2 according to Example 1 of the present invention.

[0035] FIG. 7 shows the gel electrophoresis results of step 3 according to Example 1 of the present invention.

[0036] FIG8 shows a UPLC result diagram of step 3 according to Example 1 of the present invention.

[0037] FIG. 9 shows the gel electrophoresis results according to Example 2 of the present invention.

[0038] FIG10 shows a graph showing the UPLC results according to Example 2 of the present invention.

[0039] FIG. 11 shows the gel electrophoresis results according to Example 3 of the present invention.

[0040] FIG. 12 shows a graph showing the UPLC results according to Example 3 of the present invention.

[0041] FIG13 shows a UPLC result diagram of step 1 according to Example 4 of the present invention.

[0042] FIG14 shows a UPLC result diagram of step 3 according to Example 4 of the present invention.

[0043] FIG15 shows a graph showing the UPLC results according to Example 5 of the present invention.

[0044] FIG. 16 shows a graph showing the UPLC results according to Example 6 of the present invention. DETAILED DESCRIPTION

[0045] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0046] As mentioned in the background art, the methods for synthesizing gRNA in the prior art have low purity, low production efficiency, difficulty in product purification and high cost, which makes it difficult to meet the requirements of industrial large-scale production. In this application, the inventors attempt to develop a biosynthetic method for gRNA, and thus propose a series of protection schemes of this application.

[0047] In a first typical embodiment of the present application, a method for the biosynthesis of gRNA is provided, which comprises: using RNA ligase to connect the 3′ end and the 5′ end of different substrates to form a phosphodiester bond to form a gRNA, wherein the gRNA includes a natural gRNA or a non-natural gRNA, and the number of substrates is ≥2; the connection site of the substrate is located at any one or more positions in the complementary region of the stem-loop structure, the annular region of the stem-loop structure, or the connection region between the stem-loop structures of the gRNA, and base complementary pairing can be performed within the substrate or between the substrates to form the secondary structure of the gRNA; the RNA ligase comprises: any one or more enzymes of RNA ligase family 1, 2 or 3.

[0048] In a preferred embodiment, the RNA ligase comprises one or more of the RNA ligases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24; or The RNA ligase shown in any one of NO: 24 has an enzyme with 70% or more identity, preferably 70% or more identity, 75% or more identity, 80% or more identity, 90% or more identity, 95% or more identity, 99% or more identity, 99.5% or more identity, or 99.9% or more identity.

[0049] Utilizing the above-mentioned RNA ligase, it is possible to catalyze the connection between the 3′ hydroxyl group and the 5′ phosphate group of the substrate to form a phosphodiester bond, thereby preparing a natural gRNA or a non-natural gRNA. In the present application, a natural gRNA is a gRNA composed of unmodified natural nucleotides A, C, G and U; a non-natural gRNA is a gRNA containing non-natural nucleotides. Utilizing the above-mentioned RNA ligase, natural nucleic acids can be connected, and non-natural nucleic acids can also be connected. The nicks of the above-mentioned connection can be in the stem-loop structure of the gRNA (including the stem-loop structure complementary region and the stem-loop structure annular region) and / or the connection region between the stem-loop structures, and the above-mentioned RNA ligase can all exert connection activity. The above-mentioned substrates have sequences that can perform base complementary pairing with each other or the substrates themselves, and base complementary pairing can spontaneously occur before, during or after connection using the RNA ligase, thereby generating a spatial structure and forming a gRNA with a secondary structure.

[0050] RNA ligases include:

[0051] Ligase-1, SEQ ID NO: 1, Bacteriophage T4, RNA ligase 1 (RNA ligase family 1, Rnl1):

[0052] Ligase ligase-2, SEQ ID NO: 2, Salmonella phage STP4-a, RNA ligase 2 (RNA ligase family 2, Rnl2):

[0053] Ligase-3, SEQ ID NO: 3, Bacreriophage T4, RNA ligase 2:

[0054] Ligase-4, SEQ ID NO: 4, Vibrio phage KVP40, RNA ligase 2:

[0055] Ligase ligase-5, SEQ ID NO: 5, Pyrococcus abyssi, RNA ligase 3 (RNA ligase family 3, Rnl3):

[0056] Ligase-6, SEQ ID NO: 6, Vibrio phage nt-1, RNA ligase 2:

[0057] Ligase-7, SEQ ID NO: 7, Shigella phage Sf22, RNA ligase 1:

[0058] Ligase-8, SEQ ID NO: 8, Vibrio phage VH7D, RNA ligase 1:

[0059] Ligase-9, SEQ ID NO: 9, Escherichia phage dhaeg, RNA ligase 2:

[0060] Ligase-10, SEQ ID NO: 10, Klebsiella phage KP27, RNA ligase 2:

[0061] Ligase-11, SEQ ID NO: 11, Vibrio phage V05, RNA ligase 1:

[0062] Ligase-12, SEQ ID NO: 12, Escherichia phage AR1, RNA ligase 2:

[0063] Ligase-13, SEQ ID NO: 13, Bacteriophage RM378, RNA ligase 1:

[0064] Ligase-14, SEQ ID NO: 14, Citrobacter phage CkP1, RNA ligase 1:

[0065] Ligase-15, SEQ ID NO: 15, Escherichia phage JN02, RNA ligase 1:

[0066] Ligase-16, SEQ ID NO: 16, Klebsiella phage KP179, RNA ligase 1:

[0067] Ligase-17, SEQ ID NO: 17, Vibrio phage nt-1, RNA ligase 1:

[0068] Ligase-18, SEQ ID NO: 18, Vibrio phage VH7D, RNA ligase 2:

[0069] Ligase-19, SEQ ID NO: 19, Klebsiella phage KP15, RNA ligase 2:

[0070] Ligase-20, SEQ ID NO: 20, Escherichia phage JN02, RNA ligase 2:

[0071] Ligase-21, SEQ ID NO: 21, Escherichia phage JS98, RNA ligase 2:

[0072] Ligase-22, SEQ ID NO: 22, Yersinia phage fPS-2, RNA ligase 2:

[0073] Ligase-23, SEQ ID NO: 23, Thermococcus barophilus, RNA ligase 1:

[0074] Ligase-24, SEQ ID NO: 24, Vibrio phage ValKK3, RNA ligase 2:

[0075] Identity as used herein refers to the "identity" between amino acid sequences or nucleic acid sequences, i.e., the total ratio of identical amino acid residues or nucleotides in an amino acid sequence or nucleic acid sequence. The identity of amino acid sequences or nucleic acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0076] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) identity and the same function, their active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as the proteins provided by the above sequences.

[0077] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0078] Generally speaking, according to the rules of substitution, replacement, etc., amino acids with similar properties will have similar effects when substituted with each other. For example, in the above protein, conservative amino acid substitutions may occur. "Conservative amino acid substitutions" include but are not limited to:

[0079] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0080] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

[0081] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0082] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0083] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0084] In a preferred embodiment, the substrate comprises 2 or more;

[0085] In a preferred embodiment, the length of the gRNA is 67-142 nt.

[0086] In a preferred embodiment, the length of the gRNA is 91-104 or 130-146 nt.

[0087] The length of the gRNA is 67-142 nt, including but not limited to 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 6, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, or 142 nt.

[0088] The gRNA includes crRNA and tracrRNA, wherein the length of crRNA is 17-24 nt, preferably 20 nt; the length of tracrRNA is 50-121 nt, preferably 80 nt.

[0089] The number of substrates includes but is not limited to 2, 3, 4, 5 or more, and gRNA can be prepared using the above-mentioned biosynthesis method.

[0090] In a preferred embodiment, the sequence of the gRNA includes the nucleotide sequence shown in any one of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 43.

[0091] In the present application embodiment, the gRNA synthesized is a 100nt sequence used in a commonly used CRISPR-Cas9 system derived from Streptococcus pyogenes and a 142nt sequence used in a CRISPR-Cas9 system derived from Neisseria meningitidis. The gRNAs of CRISPR-Cas9 systems from different sources all have similar secondary structures as shown in Figure 2 (Fuchsbauer et al., 2019; Hirano et al., 2016; Hirano et al., 2019; Hsu et al., 2013; Ran et al., 2015; Yamada et al., 2017). Using the above-mentioned gRNA biosynthesis method, gRNAs suitable for other CRISPR-Cas9 systems can also be synthesized.

[0092] The gRNA sequence includes a 15-25nt variable sequence, in which the base N is independently selected from any one of A, U, G, or C. This can be flexibly changed according to the crRNA required for gene editing, thereby achieving targeted editing of different target fragments. Other bases (non-N bases) in the gRNA sequence can also be designed and changed according to the preferences of CRISPR and Cas proteins.

[0093] In a preferred embodiment, the substrate comprises SEQ ID NO:29 or a nucleotide sequence consisting of 15-25 N bases; and the nucleotide sequences shown in SEQ ID NO:30 and SEQ ID NO:31; or a sequence consisting of a nucleotide sequence consisting of 15-25 N bases and a nucleotide sequence shown in SEQ ID NO:36, or a nucleotide sequence shown in SEQ ID NO:32, and a nucleotide sequence shown in SEQ ID NO:33; a nucleotide sequence consisting of 15-25 N bases located 5' to SEQ ID NO:36; or a sequence consisting of a nucleotide sequence consisting of 15-25 N bases and a nucleotide sequence shown in SEQ ID NO:47, or a nucleotide sequence shown in SEQ ID NO:44, and a nucleotide sequence shown in SEQ ID NO:45 and SEQ ID NO:46; a nucleotide sequence consisting of 15-25 N bases located 5' to SEQ ID NO:36; or a sequence consisting of a nucleotide sequence consisting of 15-25 N bases and a nucleotide sequence shown in SEQ ID NO:50, or a nucleotide sequence shown in SEQ ID NO:48, and a nucleotide sequence shown in SEQ ID NO:59. The nucleotide sequence shown in NO: 49; the nucleotide sequence consisting of 15-25 N bases is located in the 5' direction of SEQ ID NO: 50.

[0094] The variable sequence (base N) in the sequence of the above substrate can be flexibly changed as described above.

[0095] In a preferred embodiment, when there are two substrates, the biosynthesis method includes: mixing the substrates and RNA ligase, where the substrates include a first substrate and a second substrate, and the RNA ligase connects the phosphate group at the 5' end of the first substrate and the hydroxyl group at the 3' end of the second substrate to form a phosphodiester bond to obtain gRNA.

[0096] The length of the first substrate and the second substrate can be flexibly changed, including but not limited to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135 or 137 nt, as long as the sum of the lengths of the first substrate and the second substrate is 67-142 nt.

[0097] In a preferred embodiment, the 5' end of the second substrate is a 5' protecting group, and the 3' end of the first substrate is a 3' protecting group.

[0098] In a preferred embodiment, when there are three substrates, the substrates include a first substrate, a second substrate and a third substrate, and the biosynthesis method includes: mixing the first substrate, the second substrate and RNA ligase, the 5' end of the first substrate is a phosphate group, the 3' end of the first substrate is a 3' protecting group, and the 5' end and 3' end of the second substrate are hydroxyl groups, RNA ligase connects the phosphate group at the 5' end of the first substrate and the hydroxyl group at the 3' end of the second substrate to form a phosphodiester bond, thereby obtaining an intermediate product with a hydroxyl group at the 5' end; 5' phosphorylation of the intermediate product to obtain a 5' phosphorylated intermediate product; mixing the 5' phosphorylated intermediate product, the third substrate and RNA ligase, connecting the phosphate group at the 5' end of the 5' phosphorylated intermediate product and the hydroxyl group at the 3' end of the third substrate to form a phosphodiester bond, thereby obtaining gRNA; wherein the 5' end of the third substrate is a 5' protecting group.

[0099] In a preferred embodiment, the biosynthesis method is repeatedly used to connect and 5' phosphorylate four or more substrates multiple times, with two substrates connected each time, and the gRNA is obtained by connecting in sequence.

[0100] Using this method, multiple substrates can be gradually linked to form gRNA through segmented ligation, preventing the mixing of multiple substrates and disordered ligation between the substrates. The 5′ end of the intermediate product is initially a hydroxyl group, which can ensure that the substrate does not self-ligate during the preparation of the intermediate product. After the intermediate product is prepared, it is 5′ phosphorylated using, but not limited to, polynucleotide kinase to achieve the next step of ligation. After 5′ phosphorylation and before RNA ligation, the 5′ phosphorylase needs to be inactivated.

[0101] In a preferred embodiment, a template nucleic acid chain is used to guide the connection of substrates; the template nucleic acid chain has at least three base-specific bindings to different substrates, and the bases of the substrates specifically bound to the template nucleic acid chain are adjacent and form a nick, thereby obtaining a double-stranded nucleic acid structure containing the nick, wherein the adjacent bases at both ends of the nick are the 5′ end and 3′ end of different substrates, respectively, the 5′ end is a 5′ phosphate group, and the 3′ end is a hydroxyl group; RNA ligase is used to connect the 5′ phosphate and hydroxyl groups upstream and downstream of the nick to form a phosphodiester bond to obtain a double-stranded nucleic acid structure; DNA enzyme is used to digest the template nucleic acid chain on the double-stranded nucleic acid structure to obtain gRNA.

[0102] In a preferred embodiment, the sequence of the template nucleic acid chain includes any one of the nucleotide sequences shown in SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID NO: 51;

[0103] In a preferred embodiment, after the template nucleic acid chain is mixed with different substrates, it is incubated at high temperature and then slowly cooled and annealed or incubated at a constant temperature to obtain a double-stranded nucleic acid structure containing a nick; the temperature of the constant temperature incubation includes 4 to 37°C, and the constant temperature incubation time is 10 minutes or more; the slow cooling and annealing after high temperature incubation includes: incubation at 95°C for 2 minutes, cooling to 12°C at 1.2°C / min, incubation at 12°C for 10 minutes, and then cooling to 4°C.

[0104] In a preferred embodiment, the 3′ end of the substrate located at the 3′ end of the gRNA is a 3′ protecting group, and the 5′ end of the substrate located at the 5′ end of the gRNA is a 5′ protecting group.

[0105] The template nucleic acid chain is a single-stranded DNA chain composed of deoxyribonucleotides. The substrates are all capable of base-pairing with the template nucleic acid chain. Therefore, in the biosynthesis method described above, the template nucleic acid chain can guide the correct arrangement of the substrates and prevent incorrect ligation between substrates. The template nucleic acid chain is used to guide RNA ligation. The substrate first binds to the template nucleic acid chain. After specific binding, a nick is generated between the substrates, resulting in a double-stranded nucleic acid structure with a nick. The nick is terminated at the 5′ and 3′ ends of different substrates, with the 5′ end being a 5′ phosphate and the 3′ end being a hydroxyl group. RNA ligase can be used to connect the 5′ phosphate and 3′ hydroxyl groups at both ends of the nick to form a phosphodiester bond, resulting in a double-stranded nucleic acid structure without a nick. DNA enzyme is then used to digest the template nucleic acid chain on the double-stranded nucleic acid structure, thereby obtaining a single-stranded gRNA. The single-stranded gRNA contains bases capable of base-pairing, which spontaneously undergo complementary pairing, forming a secondary structure, thereby obtaining a gRNA containing a secondary structure.

[0106] For three, four, or even more substrates, the yield decreases with each additional substrate in a multi-step ligation, taking into account the efficiency of each ligation step. However, using a template nucleic acid chain to guide the ligation of substrates (the splint method) allows a single-step reaction with essentially unchanged yield.

[0107] In a preferred embodiment, the secondary structure of the gRNA is formed by spontaneous base complementary pairing of the substrate itself or between substrates.

[0108] The spontaneous base complementary pairing mentioned above can be formed spontaneously at room temperature or 4°C. Slow cooling and annealing after high temperature incubation is more conducive to the formation of secondary structure.

[0109] In a preferred embodiment, the 3′ protecting groups include GalNac (N-acetylgalactosamine group), phosphate group, -O-NH2, azido group (-CN=N + =N -, 3′-O-azidomethyl), cyanoethyl (-C-CN, 3′-O-2-cyanoethyl), allyl (-CC=C, 3′-O-allyl), 2-nitrobenzyl (3′-O-2-nitrobenzyl) and other modification groups that can prevent the formation of phosphate bonds; 5′ protecting groups include hydroxyl, hydrogen or methyl and other modification groups that can prevent the formation of phosphate bonds.

[0110] The 3'-protecting group need not be a hydroxyl group, and the 5'-protecting group need not be a phosphate group. For substrates with a 3'-terminal hydroxyl group, the 5'-terminus should not be a phosphate group; for substrates with a 5'-terminal phosphate group, the 3'-terminus should not be a hydroxyl group. This is to prevent the substrates from self-cyclizing, thereby affecting the yield and product purity of the biosynthesis method. This method can also be used to prepare non-natural RNA containing modifications.

[0111] In a preferred embodiment, the substrate includes one or more non-natural nucleotides, and the non-natural nucleotides include: ribonucleotides having one or more of ribose 2' position modification, ribose backbone modification, base modification or phosphate backbone modification; ribose 2' position modification includes 2'-methoxy modification, 2'-fluorine modification, 2'-hydrogen modification, 2'-methoxyethyl modification, 2'-FANA modification, locked nucleic acid modification or hexitol nucleic acid modification; ribose backbone modification includes replacing the ribose in the nucleotide with ribuloNA, TNA, tPhoNA, or dXNA; base modification includes deazaadenine C7 modification, deazaguanosine C7 modification, cytosine C5 modification, and uridine C5 modification; phosphate backbone modification includes PS modification.

[0112] Non-natural nucleic acids (xeno-nucleic acids, XNAs) are a class of nucleic acid molecules with non-natural backbones or nucleobases. Non-natural ribonucleotides are ribonucleotides with non-natural backbones or nucleobases. The aforementioned substrates contain non-natural nucleic acids, and the gRNA prepared by ligation also contains non-natural nucleic acids, thus belonging to non-natural RNA (non-natural gRNA).

[0113] Among them, the locked nucleic acid is modified to Hexitol nucleic acid modification 2′-methoxyethyl modified 2′-methoxy modified 2′ fluorine modified 2′-FANA ribuloNA is TNA tPhoNA dXNA is PS modified to In the above structures, Base represents a base. For the above modifications of non-natural nucleotides, please refer to the literature: Duffy K, Arangundy-Franklin S, Holliger P. Modified nucleic acids: replication, evolution, and next-generation therapeutics [J]. BMC Biology, 2020, 18 (1): 112. In non-natural nucleotides, the 2' modification increases the steric hindrance of 2', such as methoxy, or changes the properties of the 2' group, such as fluorine. The biosynthetic method disclosed in this application can connect 2' modified nucleic acids, so the above biosynthetic method can also be used to achieve the connection of natural nucleic acids with 2' hydroxyl groups to obtain natural gRNA.

[0114] In a second typical embodiment of the present application, a gRNA is provided, which includes a gRNA prepared using the above-mentioned biosynthesis method.

[0115] The beneficial effects of the present application will be further explained in detail below with reference to specific examples. The reaction diagrams of Examples 1, 2 and 3 are shown in FIG1 .

[0116] Example 1

[0117] ssRNA ligase connects three segments of single-stranded RNA to form a 100nt gRNA. The reaction diagram is shown in Figure 1A.

[0118] The sequence of the gRNA includes, but is not limited to, the sequence shown in SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27. In the gRNA sequence, a variable sequence of 15-25 nt is included, that is, the number of N located at the 5' end of the gRNA can be optionally any integer between 15 and 25, including 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25.

[0119] SEQ ID NO: 25:

[0120] SEQ ID NO: 26:

[0121] SEQ ID NO: 27:

[0122] The base N is a base that can be flexibly designed according to the CRISPR target fragment and can be flexibly selected as A, U, G or C.

[0123] Target synthetic sequence 1 in this example: SEQ ID NO: 28:

[0124] The 2' position of all ribonucleotides in the sequence shown in SEQ ID NO: 28 is modified with a methoxy group.

[0125] The secondary structure of SEQ ID NO: 26 is shown in FIG2 , and the secondary structure of SEQ ID NO: 28 is shown in FIG3 .

[0126] The three steam loops in Figures 2 and 3 are the stem-loop structures of gRNA, which include the stem-loop complementary region formed by base complementary pairing and the ring region of the stem-loop structure; the 20nt and 24nt guides are the variable regions composed of base N.

[0127] Step 1: The substrate g2 and g3 fragments are connected using RNA ligase.

[0128] RNA ligase was used for the ligation reaction. The reaction system was 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 100 μM g2, 100 μM g3, 200 μM ATP, and 0.2 mg / mL RNA ligase. The reaction temperature was 37°C and the reaction time was 16 h. After the reaction was completed, the reaction was terminated by incubation at 80°C for 5 min to obtain the intermediate product g2g3.

[0129] The reaction system was detected by urea denaturing polyacrylamide gel electrophoresis. The gel electrophoresis detection results are shown in Figure 4. Blank is the negative control group without adding RNA ligase. Compared with blank, the reaction system of RNA ligase ligase-1, ligase-2, ligase-3, ligase-4, ligase-5, ligase-6, ligase-7, ligase-8, ligase-9, ligase-10, ligase-11, ligase-12, ligase-13, ligase-14, ligase-15, ligase-16, ligase-17, ligase-18, ligase-19, ligase-20, and ligase-21 detected a new RNA band between 50 nt and 80 nt of the RNA marker. The molecular weight of the new band detected by liquid chromatography-mass spectrometry analysis was consistent with the theoretical molecular weight of the g2 and g3 ligation products.

[0130] The reaction system was tested by UPLC. The results are shown in FIG5 . The catalytic activity of RNA ligase detected by UPLC is shown in the following table (Table 1):

[0131] Table 1 Note: "++" indicates a product system purity of 20-40%, "+++" indicates a product system purity of 40-60% (excluding the endpoint of 40%), "++++" indicates a product system purity of 60-80% (excluding the endpoint of 60%), and "+++++" indicates a product system purity of 80-100% (excluding the endpoint of 80%). In this application, "product system purity" refers to the ratio of the target product to the total components in the reaction system (target product + remaining substrate) in the reaction system, which can be calculated by the area under the curve of different components in the liquid chromatography results.

[0132] The g2+g3 ligation reaction was performed under high substrate concentration conditions, with substrate concentrations of 500 or 700 μM. UPLC analysis of RNA ligase catalytic activity is shown in Table 2.

[0133] Table 2

[0134] In Table 2, the reaction system with a substrate concentration of 100 μM was: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 100 μM g2, 100 μM g3, 200 μM ATP, and 0.2 mg / mL RNA ligase. The reaction temperature was 37°C, the reaction time was 16 h, and the reaction was terminated by incubation at 80°C for 5 min.

[0135] The reaction system with a substrate concentration of 500 μM was: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 500 μM g2, 500 μM g3, 1000 μM ATP, and 0.2 mg / mL RNA ligase. The reaction temperature was 37°C for 16 h, and the reaction was terminated by incubation at 80°C for 5 min.

[0136] The reaction system with a substrate concentration of 700 μM was: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 700 μM g2, 700 μM g3, 1400 μM ATP, and 0.2 mg / mL RNA ligase. The reaction temperature was 37°C and the reaction time was 16 h. After the reaction was completed, the reaction was terminated by incubation at 80°C for 5 min.

[0137] Step 2: The 76nt intermediate product g2g3 is 5′ phosphorylated.

[0138] Oligonucleotides were 5′-phosphorylated using T4 Polynucleotide Kinase (T4 PNK, Vazyme, N102-01). In step 1, 0.25 U / μL T4 PNK, 1 mM ATP, and a corresponding volume of 10X T4 PNK Buffer were added. The reaction was incubated at 37°C for at least 3 h. P-g2g3 (5′-phosphorylated g2g3) was prepared after purification.

[0139] UPLC analysis of the phosphorylated products is shown in Figure 6. T4 PNK represents the sample prepared in Step 2, and control represents the negative control sample without T4 PNK. The g2g3 product peak disappears in the system with T4 PNK added, and a new peak appears to its right. LC / MS analysis confirms that the molecular weight of this new peak is identical to the theoretical molecular weight of P-g2g3.

[0140] Step 3: Ligate the g1 and P-g2g3 fragments using RNA ligase.

[0141] The reaction system was prepared as follows: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 20 μM g1, 20 μM P-g2g3, 40 μM ATP, and 0.2 mg / mL RNA ligase. The reaction temperature was 37 ° C and the reaction time was 16 h. After the reaction was completed, the reaction was terminated by incubation at 80 ° C for 5 min to obtain the product gRNA.

[0142] Part of the reaction system was tested using urea-denatured polyacrylamide gel electrophoresis. The gel electrophoresis test results are shown in Figure 7. A new RNA band was detected between 80nt and 150nt of the RNA Marker. The molecular weight of the new band detected by liquid chromatography-mass spectrometry analysis was consistent with the theoretical molecular weight of the gRNA of the g1 and P-g2g3 ligation product.

[0143] The reaction system was tested by UPLC, and the results are shown in FIG8 . The catalytic activity of RNA ligase detected by UPLC is shown in the following table (Table 3):

[0144] Table 3 Note: "+" indicates a product system purity of 0-5% (excluding the endpoint of 0%), "++" indicates a product system purity of 5-10% (excluding the endpoint of 5%), "+++" indicates a product system purity of 10-15% (excluding the endpoint of 10%), and "++++" indicates a product system purity of 15-20% (excluding the endpoint of 15%). In this application, "product system purity" refers to the ratio of the target product to the total components in the reaction system (target product + remaining substrate) in the reaction system, which can be calculated by the area under the curve of different components in the liquid chromatography results.

[0145] The sequences of g1, g2 and g3 are shown in Table 4.

[0146] Table 4

[0147] The m after A, C, G, or U represents a 2′-methoxy modification of the ribonucleotide, g1 contains 24 bases that can be designed according to the CRISPR target fragment, and the corresponding substrate sequence includes a variable sequence consisting of 24 (optionally 15-25) bases.

[0148] Example 2

[0149] RNA ligase connects two 50nt single-stranded RNA segments to form a 100nt gRNA. The reaction diagram is shown in Figure 1B.

[0150] Target synthetic sequence 1 in this example: SEQ ID NO: 28:

[0151] All ribonucleotides in the above sequence are methoxy-modified at 2'.

[0152] RNA ligase was used to carry out the g4 and g5 ligation reaction. The reaction system was 50mM Tris-HCl (pH7.5), 10mM MgCl2, 1mM DTT, 100μM g4, 100μM g5, 200μM ATP, and 0.2mg / mL RNA ligase. The reaction temperature was 37°C and the reaction time was 16h. After the reaction was completed, the reaction was terminated by incubation at 80°C for 5min to obtain the gRNA product.

[0153] The reaction system was detected by urea denaturing polyacrylamide gel electrophoresis. The gel electrophoresis detection results are shown in Figure 9. Blank is the negative control group without adding RNA ligase. Compared with blank, the reaction system of RNA ligase ligase-1, ligase-2, ligase-3, ligase-4, ligase-7, ligase-8, ligase-9, ligase-10, ligase-11, ligase-13, ligase-14, ligase-15, ligase-16, ligase-17, ligase-18, ligase-19, ligase-20, ligase-21, ligase-22, ligase-23, and ligase-24 detected a new RNA band between 80 nt and 150 nt of RNAMarker. The molecular weight of the new band detected by liquid chromatography-mass spectrometry analysis was consistent with the theoretical molecular weight of the gRNA product.

[0154] The reaction system was tested by UPLC, and the results are shown in FIG10 . The catalytic activity of RNA ligase detected by UPLC is shown in the following table (Table 5):

[0155] Table 5 Note: "+" indicates a product system purity of 0-10% (excluding the endpoint value of 0%), "++" indicates a product system purity of 10-20% (excluding the endpoint value of 10%), "+++" indicates a product system purity of 20-30% (excluding the endpoint value of 20%), "++++" indicates a product system purity of 30-40% (excluding the endpoint value of 30%), and "+++++" indicates a product system purity of 40-50% (excluding the endpoint value of 40%). In this application, "product system purity" means the ratio of the target product to the total components (target product + remaining substrate) in the reaction system, which can be calculated by the area under the curve of different components in the liquid chromatography results.

[0156] The sequences of g4 and g5 are shown in Table 6.

[0157] Table 6

[0158] Wherein, m after A, C, G or U represents a 2' methoxy modification of the ribonucleotide, g4 contains 24 bases that can be designed according to the CRISPR target fragment, and the corresponding substrate sequence includes a sequence containing a variable sequence N of 15-25 nt, and the number of Ns located at the 5' end of the substrate sequence can be any integer between 15 and 25, including 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, including but not limited to the sequence shown in SEQ ID NO: 34 or SEQ ID NO: 35; that is, g4 is a sequence formed by connecting the variable sequence of 15-25 nt and the sequence shown in SEQ ID NO: 36, and the variable sequence is located in the 5' direction of SEQ ID NO: 36.

[0159] 5'-GUUUUAGAGCUAGAAAUAGCAAGUUA-3' (SEQ ID NO: 36).

[0160] The g4+g5 ligation reaction was carried out under high substrate concentration conditions, and the UPLC analysis of RNA ligase catalytic activity is shown in Table 7.

[0161] Table 7

[0162] In Table 7, the reaction system with a substrate concentration of 100 μM was: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 100 μM g4, 100 μM g5, 200 μM ATP, and 0.2 mg / mL RNA ligase. The reaction temperature was 37°C, the reaction time was 16 h, and the reaction was terminated by incubation at 80°C for 5 min.

[0163] The reaction system with a substrate concentration of 300 μM was: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 300 μM g4, 300 μM g5, 600 μM ATP, and 0.2 mg / mL RNA ligase. The reaction temperature was 37°C and the reaction time was 16 h. After the reaction was completed, the reaction was terminated by incubation at 80°C for 5 min.

[0164] Example 3

[0165] RNA ligase, guided by template DNA, connects three segments of single-stranded RNA to form a 100nt gRNA. The reaction diagram is shown in Figure 1C.

[0166] Target synthetic sequence 1 in this example: SEQ ID NO: 28:

[0167] All ribonucleotides in the above sequence are methoxy-modified at 2'.

[0168] RNA ligase was used for ligation reaction, and the g1, P-g2 (5'-phosphorylated g2) and g3 fragments were connected under the guidance of gD (template nucleic acid chain). The reaction system was 50mM Tris-HCl (pH7.5), 10mM MgCl2, 1mM DTT, 60μM g1, 60μM P-g2, 60μM g3, 60μM gD, 180μM ATP, 0.2mg / mL RNA ligase, the reaction temperature was 16℃, the reaction time was 16h, and the reaction was terminated by incubation at 80℃ for 5min. A final concentration of 0.1U / μL DNase I was added and digested at 37℃ for 4h to remove the gD splint.

[0169] The reaction system was detected by urea denaturing polyacrylamide gel electrophoresis. The gel electrophoresis detection results are shown in Figure 11. Blank is the negative control group without adding RNA ligase, and gD is the control group with only adding template nucleic acid chain. Compared with blank, the reaction system of RNA ligase ligase-1, ligase-2, ligase-3, ligase-4, ligase-6, ligase-7, ligase-9, ligase-10, ligase-11, ligase-12, ligase-13, ligase-14, ligase-15, ligase-16, ligase-17, ligase-18, ligase-19, ligase-20, ligase-21, ligase-22, and ligase-24 detected a new RNA band between 80 nt and 150 nt of RNA Marker. The molecular weight of the new band detected by liquid chromatography-mass spectrometry analysis was consistent with the theoretical molecular weight of the gRNA product.

[0170] The reaction system was tested by UPLC. The results are shown in FIG12 . The catalytic activity of RNA ligase detected by UPLC is shown in the following table (Table 8):

[0171] Table 8 Note: "+" indicates a product system purity of 0-20% (excluding the endpoint value of 0%), "++" indicates a product system purity of 20-40% (excluding the endpoint value of 20%), "+++" indicates a product system purity of 40-60% (excluding the endpoint value of 40%), "++++" indicates a product system purity of 60-80% (excluding the endpoint value of 60%), and "+++++" indicates a product system purity of 80-100% (excluding the endpoint value of 80%). In this application, "product system purity" means the ratio of the target product to the total components (target product + remaining substrate) in the reaction system, which can be calculated by the area under the curve of different components in the liquid chromatography results.

[0172] The gD and P-g2 sequences are shown in Table 9.

[0173] Table 9

[0174] Among them, m after A, C, G or U indicates a 2' methoxy modification of the ribonucleotide, d before A, C, G or T indicates that the nucleotide is a deoxyribonucleotide (DNA), gD contains 12 bases that can be designed according to the CRISPR target fragment, based on the principle of base complementary pairing, the corresponding template nucleic acid chain includes a variable sequence of 3-25nt, and the template nucleic acid chain from 5' end to 3' end includes: a) a sequence that can complementarily pair with the 5' end of substrate g3; b) a sequence that can complementarily pair with substrate P-g2; c) a sequence that can complementarily pair with the 3' end of substrate g1. The template nucleic acid chain includes a variable sequence of 3-25 nt, that is, the number of N located at the 3' end of the template nucleic acid chain can be any integer from 3 to 25, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, including but not limited to the sequence shown in SEQ ID NO: 39 or SEQ ID NO: 40;

[0175] Example 4

[0176] ssRNA ligase connects three segments of single-stranded RNA to form a 142nt gRNA. The reaction diagram is shown in Figure 1A.

[0177] Target synthetic sequence 2 in this example: SEQ ID NO: 41:

[0178] The 2' ends of all ribonucleotides in the sequence shown in SEQ ID NO: 41 are modified with methoxy groups.

[0179] The 5' end of the sequence shown in SEQ ID NO: 41 contains a 21 nt variable sequence that can be designed according to the CRISPR target fragment. The length of this variable sequence is 15-25 nt, that is, the number of Ns at the 5' end of the gRNA can be any integer between 15 and 25, including 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. Including but not limited to the sequence shown in SEQ ID NO: 42 or SEQ ID NO: 43.

[0180] SEQ ID NO: 42:

[0181] SEQ ID NO: 43:

[0182] Step 1: The substrate Ng2 and Ng3 fragments are connected using RNA ligase.

[0183] RNA ligase was used for the ligation reaction. The reaction system was 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 50 μM Ng2, 50 μM Ng3, 200 μM ATP, and 0.2 mg / mL RNA ligase. The reaction temperature was 37°C and the reaction time was 16 h. After the reaction was completed, the reaction was terminated by incubation at 80°C for 5 min to obtain the intermediate product Ng2Ng3.

[0184] Using liquid chromatography (UPLC) analysis, the results are shown in Figure 13. A new peak was detected in the reaction system of RNA ligase ligase-1, ligase-2, ligase-3, ligase-4, ligase-5, ligase-6, ligase-7, ligase-8, ligase-9, ligase-10, ligase-11, ligase-12, ligase-13, ligase-14, ligase-15, ligase-16, ligase-17, ligase-18, ligase-19, ligase-20, and ligase-21. Mass spectrometry identification showed that the molecular weight of the peak was consistent with the theoretical molecular weight of the Ng2 and Ng3 ligation product.

[0185] Step 2: 5′ phosphorylation of the 84nt intermediate product Ng2Ng3.

[0186] The 5' end of Ng2Ng3 was phosphorylated using the method of step 2 in Example 1, and P-Ng2Ng3 (5' end phosphorylated Ng2Ng3) was prepared after purification. Mass spectrometry identification showed that the phosphorylated product had the correct molecular weight.

[0187] Step 3: Connect the Ng1 and P-Ng2Ng3 fragments using RNA ligase.

[0188] The reaction system was prepared as follows: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 20 μM Ng1, 20 μM P-Ng2Ng3, 40 μM ATP, and 0.2 mg / mL RNA ligase. The reaction temperature was 37 ° C, the reaction time was 16 h, and the reaction was terminated by incubation at 80 ° C for 5 min to obtain the product gRNA.

[0189] Using liquid chromatography (UPLC) analysis, the results are shown in Figure 14. A new peak was detected in the reaction system of RNA ligase ligase-1, ligase-2, ligase-3, ligase-4, ligase-5, ligase-6, ligase-7, ligase-8, ligase-9, ligase-10, ligase-11, ligase-12, ligase-13, ligase-14, ligase-15, ligase-16, ligase-17, ligase-18, ligase-19, ligase-20, and ligase-21. Mass spectrometry identified the peak molecular weight as consistent with the theoretical molecular weight of the product gRNA (142 nt) connected by Ng1 and P-Ng2Ng3.

[0190] The sequences of Ng1, Ng2 and Ng3 are shown in Table 10.

[0191] Table 10

[0192] The m following A, C, G, or U indicates a 2'-methoxy modification of the ribonucleotide. The 5' end of Ng1 contains 21 bases (N) that can be designed based on the CRISPR target fragment. The corresponding substrate sequence includes a variable sequence consisting of 21 bases (optionally 15-25). Specifically, Ng1 comprises a sequence formed by connecting a 15-25 nt variable sequence to the sequence shown in SEQ ID NO: 47, with the variable sequence located 5' to SEQ ID NO: 47.

[0193] Example 5

[0194] RNA ligase connects two 71nt single-stranded RNAs to form a 142nt gRNA. The reaction diagram is shown in Figure 1B.

[0195] Target synthetic sequence 2 in this example: SEQ ID NO: 41:

[0196] The 2' ends of all ribonucleotides in the sequence shown in SEQ ID NO: 41 are modified with methoxy groups.

[0197] The following RNA ligase was used for the ligation reaction of Ng4 and Ng5. The reaction system was 50mM Tris-HCl (pH7.5), 10mM MgCl2, 1mM DTT, 50μM Ng4, 50μM Ng5, 200μM ATP, and 0.2mg / mL RNA ligase. The reaction temperature was 37°C and the reaction time was 16h. After the reaction was completed, the reaction was terminated by incubation at 80°C for 5min to obtain the gRNA product.

[0198] Using liquid chromatography (UPLC) analysis, the results are shown in Figure 15. A new peak was detected in the reaction system of RNA ligase ligase-1, ligase-2, ligase-3, ligase-4, ligase-7, ligase-8, ligase-9, ligase-10, ligase-11, ligase-13, ligase-14, ligase-15, ligase-16, ligase-17, ligase-18, ligase-19, ligase-20, ligase-21, ligase-22, ligase-23, and ligase-24. Mass spectrometry identified the peak molecular weight as consistent with the theoretical molecular weight of the product gRNA (142 nt) connected by Ng4 and Ng5.

[0199] The sequences of Ng4 and Ng5 are shown in Table 11.

[0200] Table 11

[0201] The m following A, C, G, or U indicates a 2'-methoxy modification of the ribonucleotide. Ng4 contains 21 bases that can be designed based on the CRISPR target fragment, and the corresponding substrate sequence includes a variable sequence consisting of 21 bases (optionally 15-25 bases). Specifically, Ng4 comprises a sequence formed by connecting a 15-25 nt variable sequence to the sequence shown in SEQ ID NO: 50, with the variable sequence located 5' to SEQ ID NO: 50.

[0202] SEQ ID NO: 50:

[0203] Example 6

[0204] RNA ligase, guided by template DNA, connects three segments of single-stranded RNA to form a 142nt gRNA. The reaction diagram is shown in Figure 1C.

[0205] Target synthetic sequence 2 in this example: SEQ ID NO: 41:

[0206] The 2' ends of all ribonucleotides in the sequence shown in SEQ ID NO: 41 are modified with methoxy groups.

[0207] RNA ligase was used to carry out the ligation reaction, and the Ng1, P-Ng2 (5′-phosphorylated g2) and Ng3 fragments were connected under the guidance of Ng-D (template nucleic acid chain). The reaction system was 50mM Tris-HCl (pH7.5), 10mM MgCl2, 1mM DTT, 60μM Ng1, 60μM P-Ng2, 60μM Ng3, 60μM Ng-D, 180μM ATP, and 0.2mg / mL RNA ligase. The reaction temperature was 16°C and the reaction time was 16h. After the reaction was completed, the reaction was terminated by incubation at 80°C for 5min.

[0208] Using liquid chromatography (UPLC) analysis, the results are shown in Figure 16. A new peak was detected in the reaction system of RNA ligase ligase-1, ligase-2, ligase-3, ligase-4, ligase-6, ligase-7, ligase-9, ligase-10, ligase-11, ligase-12, ligase-13, ligase-14, ligase-15, ligase-16, ligase-17, ligase-18, ligase-19, ligase-20, ligase-21, ligase-22, and ligase-24. Mass spectrometry identified the peak molecular weight as consistent with the theoretical molecular weight of the product gRNA (142 nt) connected by Ng1, P-Ng2, and Ng3.

[0209] The sequences of Ng-D and P-Ng2 are shown in Table 12.

[0210] Table 12

[0211] Among them, m after A, C, G or U indicates a 2' methoxy modification of the ribonucleotide, d before A, C, G or T indicates that the nucleotide is a deoxyribonucleotide (DNA), and the template nucleic acid chain Ng-D includes from the 5' end to the 3' end: a) a sequence that can complementarily pair with the 5' end of the substrate Ng3; b) a sequence that can complementarily pair with the substrate P-Ng2; c) a sequence that can complementarily pair with the 3' end of the substrate Ng1.

[0212] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: using the above-mentioned biosynthesis method, through RNA ligases of families such as Rnl1 and Rnl2, it is possible to connect two or more substrates with or without the assistance of a template nucleic acid chain to achieve connection between substrates. The nick position of the connection can be located on the stem-loop structure of the gRNA, or in the connection region between the stem-loop structures, thereby connecting multiple substrates into a complete RNA chain, and the substrates can perform base complementary pairing on their own, thereby preparing a gRNA with a secondary structure. Compared to the solid-phase synthesis method in the prior art, the biosynthesis method disclosed in the present invention does not require a solid support, the product purity is relatively high, the product and impurity chain length are greatly different, it is easy to purify, and there are no branched impurities. Compared to the in vitro transcription method in the prior art, the product prepared by the biosynthesis method disclosed in the present invention is relatively high in purity, easy to purify, and has clear 5' and 3' ends.

[0213] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for biosynthesis of gRNA, characterized in that: The biosynthetic method comprises: Using RNA ligase to connect the 3′ end and the 5′ end of different substrates to form a phosphodiester bond to form the gRNA, wherein the gRNA includes a natural gRNA or a non-natural gRNA; The connection site of the substrate is located at any one or more positions in the stem-loop structure complementary region, the stem-loop structure ring region or the connection region between the stem-loop structures of the gRNA, Base complementary pairing can be performed inside the substrate or between the substrates to form the secondary structure of the gRNA; The RNA ligase includes any one or more enzymes of RNA ligase families 1, 2 and 3.

2. The biosynthesis method according to claim 1, characterized in that The RNA ligase comprises: one or more of the RNA ligases set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24; or An enzyme having 70% or more identity with the RNA ligase represented by any one of SEQ ID NO: 1 to SEQ ID NO:

24.

3. The biosynthesis method according to claim 1, characterized in that The substrates include 2 or more.

4. The biosynthesis method according to claim 1, characterized in that The length of the gRNA is 67-146nt.

5. The biosynthesis method according to claim 1, characterized in that The sequence of the gRNA includes the nucleotide sequence shown in any one of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO:

43.

6. The biosynthesis method according to claim 5, characterized in that The substrate includes SEQ ID NO: 29 or a nucleotide sequence consisting of 15-25 N bases; and the nucleotide sequences shown in SEQ ID NO: 30 and SEQ ID NO: 31; or A sequence formed by connecting a nucleotide sequence consisting of 15-25 N bases and the nucleotide sequence shown in SEQ ID NO: 36, or the nucleotide sequence shown in SEQ ID NO: 32, and the nucleotide sequence shown in SEQ ID NO: 33; the nucleotide sequence consisting of 15-25 N bases is located in the 5' direction of SEQ ID NO: 36; or A sequence formed by connecting a nucleotide sequence consisting of 15-25 N bases and the nucleotide sequence shown in SEQ ID NO: 47, or a nucleotide sequence shown in SEQ ID NO: 44, and a nucleotide sequence shown in SEQ ID NO: 45 and SEQ ID NO: 46; the nucleotide sequence consisting of 15-25 N bases is located at the 5' end of SEQ ID NO: 36 direction; or A sequence formed by connecting a nucleotide sequence consisting of 15-25 N bases and the nucleotide sequence shown in SEQ ID NO: 50, or the nucleotide sequence shown in SEQ ID NO: 48, and the nucleotide sequence shown in SEQ ID NO: 49; the nucleotide sequence consisting of 15-25 N bases is located in the 5' direction of SEQ ID NO:

50.

7. The biosynthesis method according to claim 3, characterized in that: When there are two substrates, the biosynthesis method comprises: The substrate and the RNA ligase are mixed, wherein the substrate includes a first substrate and a second substrate, and the RNA ligase connects the phosphate group at the 5' end of the first substrate and the hydroxyl group at the 3' end of the second substrate to form the phosphodiester bond, thereby obtaining the gRNA.

8. The biosynthesis method according to claim 7, characterized in that The 5' end of the second substrate is a 5' protecting group, and the 3' end of the first substrate is a 3' protecting group.

9. The biosynthesis method according to claim 3, characterized in that: When there are three substrates, the substrates include a first substrate, a second substrate and a third substrate, and the biosynthesis method includes: The first substrate, the second substrate and the RNA ligase are mixed, the 5′ end of the first substrate is a phosphate group, the 3′ end of the first substrate is a 3′ protecting group, and the 5′ end and 3′ end of the second substrate are hydroxyl groups, The RNA ligase connects the phosphate group at the 5′ end of the first substrate and the hydroxyl group at the 3′ end of the second substrate to form the phosphodiester bond, thereby obtaining an intermediate product with a hydroxyl group at the 5′ end; 5′-phosphorylating the intermediate product to obtain a 5′-phosphorylated intermediate product; The 5′ phosphorylation intermediate, the third substrate and the RNA ligase are mixed, and the RNA ligase connects the phosphate group at the 5′ end of the 5′ phosphorylation intermediate and the hydroxyl group at the 3′ end of the third substrate to form the phosphodiester bond, thereby obtaining the gRNA; Wherein, the 5' end of the third substrate is a 5' protecting group.

10. The biosynthesis method according to claim 9, characterized in that The biosynthesis method is repeatedly used to connect and 5′ phosphorylate four or more substrates for multiple times, with two substrates connected each time, and the gRNA is obtained by connecting in sequence.

11. The biosynthesis method according to claim 1, characterized in that: using a template nucleic acid strand to direct the ligation of the substrate; The template nucleic acid chain has at least three bases specifically bound to different substrates, the bases of the substrates specifically bound to the template nucleic acid chain are adjacent to each other and form a nick, and a double-stranded nucleic acid structure containing a nick is obtained, the bases adjacent to both ends of the nick are the 5' end and the 3' end of different substrates, the 5' end is a 5' phosphate, and the 3' end is a hydroxyl group; Using the RNA ligase to connect the 5′ phosphate and the hydroxyl groups upstream and downstream of the nick to form the phosphodiester bond and obtain a double-stranded nucleic acid structure; The template nucleic acid chain on the double-stranded nucleic acid structure is digested with DNA enzyme to obtain the gRNA.

12. The biosynthesis method according to claim 11, characterized in that: The sequence of the template nucleic acid chain includes any one of the nucleotide sequences shown in SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40 or SEQ ID NO:

51.

13. The biosynthesis method according to claim 11, characterized in that: After the template nucleic acid chain is mixed with the different substrates, the mixture is incubated at a constant temperature or at 95° C. and then cooled and annealed to obtain the double-stranded nucleic acid structure containing the nick; The constant temperature incubation temperature includes 4 to 37° C., and the constant temperature incubation time is ≥ 10 min; The cooling annealing after incubation at 95° C. includes: incubating at 95° C. for 2 minutes, cooling to 12° C. at 0.5-1.2° C. / min, and incubating at 12° C. for 10 minutes and then cooling to 4° C.

14. The biosynthesis method according to claim 11, characterized in that: The 3′ end of the substrate located at the 3′ end of the gRNA is a 3′ protecting group, and the 5′ end of the substrate located at the 5′ end of the gRNA is a 5′ protecting group.

15. The biosynthesis method according to claim 1, characterized in that: The secondary structure of the gRNA is formed by spontaneous base complementary pairing of the substrate itself or between substrates.

16. The biosynthetic method according to claim 9 or 14, characterized in that: The 3' protecting group includes N-acetylgalactosamine group, phosphate group, -O-NH2, azido group, cyanoethyl group, allyl group or 2-nitrobenzyl group; the 5' protecting group includes hydroxyl group, hydrogen or methyl group.

17. The biosynthetic method according to any one of claims 1 to 15, characterized in that The substrate includes one or more non-natural nucleotides, and the non-natural nucleotides include: ribonucleotides with one or more of ribose 2' position modification, ribose backbone modification, base modification or phosphate backbone modification; The modification of the 2′ position of ribose includes 2′-methoxy modification, 2′-fluorine modification, 2′-hydrogen modification, 2′-methoxyethyl modification, 2′-FANA modification, locked nucleic acid modification or hexitol nucleic acid modification; The ribose backbone modification includes replacing the ribose in the nucleotide with ribuloNA, TNA, tPhoNA or dXNA; The base modification includes deazaadenine C7 modification, deazaguanosine C7 modification, cytosine C5 modification or uridine C5 modification; The phosphate backbone modification includes PS modification.

18. A gRNA, characterized in that The gRNA includes the gRNA prepared by the biosynthetic method according to any one of claims 1 to 17.

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