WO2019185034A1 - 一种sgRNA的体外合成方法及其试剂盒 - Google Patents

一种sgRNA的体外合成方法及其试剂盒 Download PDF

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WO2019185034A1
WO2019185034A1 PCT/CN2019/080485 CN2019080485W WO2019185034A1 WO 2019185034 A1 WO2019185034 A1 WO 2019185034A1 CN 2019080485 W CN2019080485 W CN 2019080485W WO 2019185034 A1 WO2019185034 A1 WO 2019185034A1
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sequence
sgrna
rna polymerase
synthesis system
nucleic acid
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French (fr)
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朱化星
张清仪
石加加
赵曼曼
何翼
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上海欣百诺生物科技有限公司
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Priority to US17/043,119 priority Critical patent/US20210115437A1/en
Publication of WO2019185034A1 publication Critical patent/WO2019185034A1/zh

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Definitions

  • the present invention relates to the field of biotechnology, and in particular to an in vitro synthesis method of sgRNA and a kit thereof.
  • the CRISPR (clustered regular interspaced short palin-dromic repeats)/Cas (CRISPR-associated) system is an acquired immune system unique to bacteria and archaea. This system is specifically cleavable to degrade exogenous DNA by specific sequence RNA guidance. .
  • the CRISPR/Cas system can be divided into three types, of which the Type II CRISPR/Cas system has been transformed into a tool for genome-targeted editing due to its simple composition.
  • a sgRNA single guide RNA
  • the sgRNA guides the Cas protein to specifically cleave the DNA sequence of interest.
  • CRISPR/Cas system can achieve various purposes in research, such as cutting, modifying, silencing, knocking out, and regulating expression of genes.
  • CRISPR/Cas has become a powerful tool for gene editing and research, with a very bright and broad application prospect.
  • RNA As an indispensable sgRNA in the CRISPR/Cas system, the synthesis method is very important. How to obtain available sgRNA at a lower cost has become the focus of researchers.
  • the chemical synthesis of RNA has a time period of 7 to 14 days and is expensive and unsuitable for sgRNA synthesis.
  • the method of constructing a plasmid vector for transcribed RNA in vivo requires cumbersome and time-consuming vector construction work, and is not flexible enough, and each vector can only correspond to a small amount of specific RNA.
  • the traditional sgRNA in vitro transcription method has many cumbersome steps, usually 1. PCR amplification of template DNA, 2. Glue recovery PCR product, 3. Transcription of sgRNA using PCR product as template. Its operation takes time and effort, and the entire process takes 5-6 hours. In the meantime, a variety of instruments such as a PCR instrument, an electrophoresis tank, and a centrifuge are required, and the reagents involved include a PCR amplification enzyme, a gel recovery kit, and T 7 RNase, so the average cost is not low.
  • the invention provides a simple, convenient, time-saving, efficient and economical synthesis method of sgRNA in vitro.
  • a first aspect of the invention provides a sgRNA synthesis system comprising:
  • Y1 is the RNA polymerase initiation region
  • L1 is a no or a joining sequence
  • Y2 is the target DNA sequence
  • L2 is a no or a joining sequence
  • Y3 is a downstream primer binding region
  • Y4 is a no or nucleotide sequence
  • each "-" is independently a bond or a nucleotide linkage sequence
  • the RNA polymerase is selected from the group consisting of T7 RNA polymerase, Sp6 RNA polymerase, U6 RNA polymerase, T3 RNA polymerase, or a combination thereof.
  • the RNA polymerase promoter region is a T7 RNA polymerase promoter region.
  • sequence structure of the element Y1 is: N X -TAATACGACTCACTATA (positions 2-18 of SEQ ID NO.: 1) - G Y , where N is A, T, C or G, X An integer of 1-6, Y is an integer from 0-2.
  • sequence of the element Y1 is as shown in SEQ ID NO.: 1.
  • sequence of the element Y1 is as shown in SEQ ID NO.: 2.
  • sequence of the element Y1 is as shown in SEQ ID NO.: 3.
  • the element Y1 has a length of 17-40 bp, preferably 18-25 bp.
  • the element Y2 has a length of 0-100 bp, preferably 0-90 bp.
  • the element Y3 has a length of 5-30 bp, preferably 10-20 bp.
  • the element Y4 has a length of 5-30 bp, preferably 8-20 bp.
  • the ligation sequence is 1-30 nt in length.
  • some or all of the L2 and/or Y3 may be used as a barcode sequence.
  • the L2 and/or Y3 contain a barcode sequence.
  • the L2 is a barcode sequence.
  • the Y3 is a universal primer binding region.
  • the downstream primer comprises a specific primer, a universal primer, a barcode primer.
  • the downstream primer is used as a universal primer or a barcode primer.
  • component (d) corresponds to the RNA polymerase promoter region in component (a).
  • the sgRNA synthesis system further comprises one or more components selected from the group consisting of:
  • the substrate for the synthetic RNA comprises: a nucleoside monophosphate, a nucleoside triphosphate, or a combination thereof.
  • the substrate for the synthetic DNA comprises: deoxynucleoside monophosphate, deoxynucleoside triphosphate, or a combination thereof.
  • the DNA polymerase is selected from the group consisting of Klenow polymerase, Taq enzyme, Pfu enzyme, KOF enzyme, Bst enzyme, Phi29 enzyme, or a combination thereof.
  • the RNA polymerase is selected from the group consisting of T7 RNA polymerase, Sp6 RNA polymerase, U6 RNA polymerase, T3 RNA polymerase, or a combination thereof.
  • the magnesium ion is derived from a source of magnesium ions selected from the group consisting of magnesium chloride, magnesium acetate, magnesium glutamate, magnesium phosphate, or combinations thereof.
  • the buffer is selected from the group consisting of Tris-HCl, 4-hydroxyethylpiperazineethanesulfonic acid, trishydroxymethylaminomethane, phosphate buffer, citrate buffer, or combination.
  • the mass concentration (wt%) of the component (a) in the sgRNA synthesis system is from 0.0008% to 0.006%, preferably from 0.001% to 0.005%, more preferably from 0.0013%. 0.00438%, based on the total weight of the sgRNA synthesis system.
  • the molar concentration (mol/L) of component (a) in the sgRNA synthesis system is 0.3 umol/L to 2 umol/L, preferably 0.5 umol/L to 1.5 umol/L. More preferably, 0.8 umol / L - 1.2 umol / L, based on the total volume of the sgRNA synthesis system.
  • the mass concentration (wt%) of the component (b) in the sgRNA synthesis system is 0.00024% to 0.0011%, preferably 0.0003% to 0.00093%, more preferably 0.00039% - 0.00081% based on the total weight of the sgRNA synthesis system.
  • the enzyme activity (U/uL) of component (c) in the sgRNA synthesis system is 0.05 U/uL-0.4 U/uL, preferably 0.08 U/uL-0.25 U/ uL, more preferably, 0.1 U/uL-0.2 U/uL, based on the total volume of the sgRNA synthesis system.
  • the enzyme activity (U/uL) of component (d) in the sgRNA synthesis system is 1 U/uL-6 U/uL, preferably 2 U/uL-5 U/uL, more preferably Ground, 3 U/uL-4 U/uL, based on the total volume of the sgRNA synthesis system.
  • the molar concentration (mol/L) of the component (e) in the sgRNA synthesis system is 0.1 mmol/L to 3 mmol/L, preferably 0.3 mmol/L to 2 mmol/L. More preferably, 0.5 mmol/L to 1.5 mmol/L, based on the total volume of the sgRNA synthesis system.
  • the molar concentration (mol/L) of the component (f) in the sgRNA synthesis system is 0.8 mmol/L to 4 mmol/L, preferably 1.2 mmol/L to 3 mmol/L. More preferably, 1.5 mmol/L to 2.5 mmol/L, based on the total weight of the sgRNA synthesis system.
  • the volume concentration (v/v) of the component (g) in the sgRNA synthesis system is from 15% to 35%, preferably from 18% to 32%, more preferably, from 20%. -30% based on the total volume of the sgRNA synthesis system.
  • the volume concentration (v/v) of the component (j) in the sgRNA synthesis system is from 5% to 15%, preferably from 6% to 13%, more preferably, 8%. -11% based on the total volume of the sgRNA synthesis system.
  • the volume concentration (v/v) of the component (1) in the sgRNA synthesis system is 0.2% to 0.7%, preferably 0.3% to 0.6%, more preferably 0.4%. -0.6% based on the total weight of the sgRNA synthesis system.
  • the molar concentration (mol/L) of the component (m) in the sgRNA synthesis system is from 1 mmol/L to 1.5 mmol/L, preferably from 1.1 mmol/L to 1.4 mmol/L. More preferably, 1.2 mmol/L to 1.3 mmol/L, based on the total weight of the sgRNA synthesis system.
  • the molar concentration (mol/L) of the component (n) in the sgRNA synthesis system is from 0.3 mmol/L to 0.7 mmol/L, preferably from 0.35 mmol/L to 0.6 mmol/ L, more preferably, 0.4 mmol/L to 0.5 mmol/L, based on the total weight of the sgRNA synthesis system.
  • the component (m) / component (n) is 10:1 to 4:1, preferably 8:1 to 3:1, more preferably 5:1. :1.
  • the molar concentration (mol/L) of the component (o) in the sgRNA synthesis system is 3.5 mmol/L to 7 mmol/L, preferably, 4 mmol/L to 6.5 mmol/L. More preferably, 5 mmol/L to 6 mmol/L is based on the total weight of the sgRNA synthesis system.
  • the sgRNA synthesis system has the following properties:
  • the total amount of sgRNA synthesis is > 1 ug (1-10 ug, preferably 1-5 ug).
  • a second aspect of the invention provides a method of synthesizing sgRNA in vitro comprising the steps of:
  • step (ii) incubated the synthetic system of step (i) for a period of time T1 under suitable conditions to synthesize the sgRNA.
  • the method further comprises: (iii) isolating or detecting the sgRNA, optionally from the sgRNA synthesis system.
  • the reaction temperature is from 25 ° C to 42 ° C, preferably from 35 ° C to 40 ° C.
  • the reaction time T1 is from 0.5 h to 8 h, preferably from 0.5 to 3 h, preferably from 0.8 h to 1 h.
  • a third aspect of the invention provides a kit for sgRNA synthesis, comprising:
  • nucleic acid construct (k1) a first container, and a nucleic acid construct located in the first container, the nucleic acid construct having the structure of formula I of 5'-3':
  • Y1 is the RNA polymerase initiation region
  • L1 is a no or a joining sequence
  • Y2 is the target DNA sequence
  • L2 is a no or a joining sequence
  • Y3 is a downstream primer binding region
  • Y4 is a no or nucleotide sequence
  • each "-" is independently a bond or a nucleotide linkage sequence
  • first container and the second container are the same container or different containers.
  • the kit further comprises an optional one or more containers selected from the group consisting of:
  • a fourth aspect of the invention provides a nucleic acid construct having the structure of formula I of 5'-3':
  • Y1 is the RNA polymerase initiation region
  • L1 is a no or a joining sequence
  • Y2 is the target DNA sequence
  • L2 is a no or a joining sequence
  • Y3 is a downstream primer binding region
  • Y4 is a no or nucleotide sequence
  • each "-" is independently a bond or nucleotide linkage sequence.
  • the RNA polymerase is selected from the group consisting of T7 RNA polymerase, Sp6 RNA polymerase, U6 RNA polymerase, T3 RNA polymerase, or a combination thereof.
  • the RNA polymerase promoter region is a T7 RNA polymerase promoter region.
  • sequence structure of the element Y1 is: N X -TAATACGACTCACTATA (positions 2-18 of SEQ ID NO.: 1) - G Y , where N is A, T, C or G, X An integer of 1-6, Y is an integer from 0-2.
  • sequence of the element Y1 is as shown in SEQ ID NO.: 1.
  • sequence of the element Y1 is as shown in SEQ ID NO.: 2.
  • sequence of the element Y1 is as shown in SEQ ID NO.: 3.
  • the element Y1 has a length of 17-40 bp, preferably 18-25 bp.
  • the element Y2 has a length of 0-100 bp, preferably 0-90 bp.
  • the element Y3 has a length of 5-30 bp, preferably 10-20 bp.
  • the element Y4 has a length of 5-30 bp, preferably 8-20 bp.
  • the ligation sequence is 1-30 nt in length.
  • some or all of the L2 and/or Y3 may be used as a barcode sequence.
  • the L2 and/or Y3 contain a barcode sequence.
  • the L2 is a barcode sequence.
  • the Y3 is a universal primer binding region.
  • Figure 1 shows the in vitro transcriptional synthesis of sgRNA electropherograms of the A and B protocols.
  • a and B respectively represent the experimental systems of schemes A and B; lane 1 is Marker; lanes 2-5 are reaction durations of 0.5 h, 1 h, 1.5 h, and 2 h, respectively.
  • Figure 2 shows the in vitro transcriptional synthesis of sgRNA electropherograms of C and D protocols.
  • C and D respectively represent the experimental system of C and D;
  • Lane 1 is Marker;
  • lanes 2-5 are 0.5h, 1h, 1.5h, and 2h reaction time respectively.
  • Figure 3 shows the electropherogram of in vitro transcription synthesis of sgRNA in the E protocol.
  • Lane 5 is Marker; lanes 1-4 are reaction durations of 0.5 h, 1 h, 1.5 h, and 2 h, respectively.
  • Figure 4 shows an in vitro transcriptional contrast electrophoresis map of the F protocol and the C protocol sgRNA.
  • Figure 5 shows that positive indicates that the positive control sgRNA is added to the CRISPR/Cas cleavage system
  • MK is DNA Ladder 2000
  • A1, A2, A3, and A4 represent the sgRNA obtained by adding four reaction durations in the A program in the CRISPR/Cas cutting system, respectively. That is, 0.5h, 1h, 1.5h, 2h; B1-4, C1-4, D1-4 and so on are the sgRNAs obtained by adding four reaction periods of B, C, and D schemes; F1 and F2 are F-program sgRNA amplification. The duration is 3h and 2h respectively.
  • the sgRNA synthesis system of the present invention can significantly increase the synthesis yield of sgRNA, the yield is ⁇ 1 ug (1-10 ug, preferably 1-5 ug), and the entire process time can be greatly shortened (for example, to less than 1 hour). The steps are simple and the average cost is also greatly reduced (about 50% down). On the basis of this, the inventors completed the present invention.
  • the invention provides a nucleic acid construct by using an RNA polymerase promoter region (such as a T7 RNA polymerase promoter region), a target DNA sequence, a downstream primer binding region (such as a specific primer, a universal primer, a barcode primer or can be
  • RNA polymerase promoter region such as a T7 RNA polymerase promoter region
  • a target DNA sequence such as a T7 RNA polymerase promoter region
  • a downstream primer binding region such as a specific primer, a universal primer, a barcode primer or can be
  • the downstream primers used as universal primers, barcode primers, and optionally additional nucleotide sequences are joined by ligation sequences, making sgRNA synthesis in vitro simple, convenient, time-saving, and efficient at a low cost.
  • the construct of the invention is as described in the first aspect of the invention.
  • the sequence structure of the RNA polymerase promoter region is: N X -TAATACGACTCACTATA (positions 2-18 of SEQ ID NO.: 1)-G Y , wherein N is A, T, C or G, and X is An integer of 1-6, Y is an integer from 0-2.
  • RNA polymerase promoter region is the T7 polymerase promoter region and the sequence is set forth in SEQ ID NO.: 1.
  • RNA polymerase promoter region is the SP6 polymerase promoter region and the sequence is set forth in SEQ ID NO.: 2.
  • RNA polymerase promoter region is the T3 polymerase promoter region and the sequence is set forth in SEQ ID NO.: 3.
  • the invention provides an in vitro sgRNA synthesis system comprising:
  • the in vitro sgRNA synthesis system further comprises:
  • the downstream primer is not particularly limited and may include a universal primer or a barcode primer, or may be used as a universal primer or a barcode primer.
  • the concentration of the downstream primer is 1 uM.
  • the RNA polymerase is not particularly limited and may be selected from one or more RNA polymerases, and a typical RNA polymerase is T7 RNA polymerase. Typically, the concentration of RNA polymerase is 3.5 U/ul.
  • the DNA polymerase is not particularly limited and may be selected from one or more DNA polymerases including, but not limited to, Klenow polymerase, Taq enzyme, Pfu enzyme, KOF enzyme, Bst enzyme, and / Or Phi29 enzyme.
  • a typical DNA polymerase is the Taq enzyme.
  • the concentration of DNA polymerase is 0.15 U/ul.
  • the mixture of nucleoside triphosphates in the sgRNA synthesis system is adenine nucleoside triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine nucleoside triphosphate.
  • the concentration of each of the single nucleotides is not particularly limited, and usually the concentration of each single nucleotide is from 0.1 mM to 0.5 mM, preferably from 0.25 mM to 0.35 mM.
  • the mixture of deoxynucleoside triphosphates in the sgRNA synthesis system is adenine deoxynucleoside triphosphate, guanine deoxynucleoside triphosphate, cytosine deoxynucleoside triphosphate, and thymidine triphosphate .
  • the concentration of each of the monodeoxynucleotides is not particularly limited, and usually the concentration of each of the monodeoxynucleotides is from 0.05 mM to 0.15 mM, preferably from 0.1 mM to 0.13 mM.
  • the concentration of the components (e) to (p) is not particularly limited, and the synthetic sgRNA can be used in the usual concentrations of the components (e) to (p).
  • the sgRNA synthesis system comprises:
  • kits for sgRNA synthesis comprising:
  • (k1) a first container, and the nucleic acid construct of the first aspect of the invention located in the first container;
  • first container and the second container are the same container or different containers.
  • a particularly preferred kit for in vitro sgRNA synthesis comprises an in vitro sgRNA synthesis system comprising: Tris-HCl 40 mM, MgCl 2 6 mM, DTT 1 mM, spermidine 2 mM, A component 1 uM, B component 1 uM, dNTPs 0.5 mM, NTPs 1.25 mM, T7 RNA polymerase 3.5 U/ul, Taq DNA polymerase 0.15 U/ul.
  • the present invention is the first to develop a simple, convenient, time-saving, efficient and economical synthesis method of sgRNA in vitro.
  • the method of the present invention can greatly reduce the entire process time (e.g., to less than 1 hour), the steps are simple, and the average cost is also greatly reduced (about 50%).
  • the method of the present invention removes the PCR amplification and gel recovery steps of the conventional RNA in vitro transcription method, and reduces the duration of the entire process from 4 to 6 hours to 1 hour.
  • the invention also reduces the amount of reagents that need to be added, making the entire operation easier.
  • the method of the present invention eliminates the conventional transcription method after the PCR amplification is omitted, and the one-step yield exceeds the conventional RNA in vitro transcription method in the case of reducing the total duration.
  • the yield is ⁇ 1 ug (1-10 ug, preferably 1-5 ug).
  • the sgRNA is transcribed in vitro for a reaction buffer for the synthesis reaction, and its specific components are: Tris-HCl, MgCl 2 , DTT, spermidine, sgRNA-R, dNTPs, NTP.
  • the Buffer is specifically:
  • an Enzyme Mix for sgRNA in vitro one-step transcription synthesis reaction is a mixture of two enzymes, one is DNA polymerase (Klenow polymerase, Taq enzyme, Pfu enzyme, KOF enzyme, Bst enzyme) , the Phi29 of one enzyme), a second RNA polymerase (T 7 enzyme, Sp6 enzymes, one enzyme of U6).
  • Enzyme Mix Taq enzyme is combined with T 7 RNA polymerase.
  • the forward (F) primer design was carried out by selecting the 5' end 20 bp of the Target DNA sequence PAM (NGG).
  • the primer structure consisted of T7promoter (20 bp), Target DNA fragment (20 bp) and the fragment actually combined with the reverse primer (10-25 bp). .
  • the schematic of the primer design is as follows:
  • Target DNA fragment For example, suppose the Target DNA fragment is a
  • the forward primers were designed and synthesized according to the design requirements of the primers, and were prepared according to the following system and reacted at 37 ° C for 1 h:
  • Example 1 Five in vitro transcription systems of A, B, C, D, and E were designed, and F was designed as a traditional in vitro transcription system, and the advantages and disadvantages of the comparison schemes were compared.
  • sgRNA-F sgRNA forward primer
  • sgRNA-R sgRNA reverse primer
  • the sgRNA-F and sgRNA-R were dissolved in RNase Free Water to prepare a concentration of 5 uM.
  • reaction buffer 1 1. The experimental system of A and B schemes, mixing all the components except the primers, and preparing into reaction buffer 1, the components of which are as follows:
  • the A and B protocols were prepared as follows: 20 ⁇ L of the reaction system:
  • each set was repeated 4 times, placed in a PCR instrument, and reacted at 37 ° C.
  • the reaction durations of the four replicates were 0.5 h, 1 h, 1.5 h, and 2 h, respectively.
  • 1 ⁇ l of DNase I was added and reacted at 37 ° C for 10 min to remove the DNA template, and the product was subjected to electrophoresis. The results are shown in Fig. 1.
  • reaction buffer 2 C and D program experimental system, in addition to the forward primer and other components other than the two enzymes, formulated into reaction buffer 2, the components are as follows:
  • the Taq enzyme and the T7 RNase are mixed together to form an Enzyme Mix:
  • each set was repeated 4 times, placed in a PCR instrument, and reacted at 37 ° C.
  • the reaction durations of the four replicates were 0.5 h, 1 h, 1.5 h, and 2 h, respectively.
  • 1 ⁇ l of DNase I was added and reacted at 37 ° C for 10 min to remove the DNA template, and the product was subjected to electrophoresis. The results are shown in Fig. 2.
  • the E protocol prepared a 20 ⁇ L reaction system as follows:
  • the forward primer, reverse primer and Template Amplification Mix were formulated into the following 20 ⁇ L PCR reaction system:
  • the reaction system was placed in a PCR machine, and PCR amplification was carried out according to the following procedure:
  • the PCR amplification product was formulated into the following 20 ⁇ L reaction system:
  • the reaction was carried out at 37 ° C for 2 hours, and the results of sgRNA synthesis were compared with the C scheme as shown in FIG. 4 .
  • the optimal effect of the scheme C was the highest, and the highest yield of the synthesized sgRNA was 1ug-2ug. And unexpectedly, its yield is much higher than the traditional sgRNA synthesis method (the sgRNA yield of the traditional synthesis method is about 100 ng-200 ng), which is 5-10 times higher.
  • the results show that when the NTP/dNTP in the reaction system is 10:1 to 4:1, preferably 8:1 to 3:1, more preferably 5:1 to 2:1, the sgRNA synthesized by the present invention It has the activity of guiding Cas9 to cleave a specific site.
  • the sgRNA obtained in addition to the D-scheme 0.5 h transcription time was too small to guide the Cas9 protein to cleave DNA, and all other DNA was cleaved by the sgRNA-directed Cas9 protein.
  • the sgRNA synthesized by the one-step in vitro transcription method of the present invention has an activity of guiding the Cas9 cleavage specific site.

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Abstract

本发明提供了一种sgRNA的体外合成方法及其试剂盒，具体地，本发明提供了具有5'-3'的式I所示的结构的核酸构建物：Y1-L1-Y2-L2-Y3-Y4 (I)，其中，Y1为RNA聚合酶启动区；L1为无或连接序列；Y2为靶DNA序列；L2为无或连接序列；Y3为下游引物结合区；Y4为无或核苷酸序列；并且，各"-"独立地为键或核苷酸连接序列。

Description

一种sgRNA的体外合成方法及其试剂盒 技术领域
本发明涉及生物技术领域，具体地，涉及一种sgRNA的体外合成方法及其试剂盒。
背景技术
CRISPR(clustered regularly interspaced short palin-dromic repeats)/Cas(CRISPR-associated)系统是一些细菌及古细菌中特有的获得性免疫系统，这个系统通过特定序列的RNA引导，特异性切割降解外源性DNA。CRISPR/Cas系统可分为三种类型,其中II型CRISPR/Cas系统由于其组成简单，被改造成为基因组靶向编辑的工具。而通过人工设计并在体外转录RNA，可以合成具有引导作用的sgRNA(single guide RNA)，sgRNA引导Cas蛋白特异性切割目的DNA序列。通过对Cas蛋白的改造，在sgRNA的引导下，CRISPR/Cas系统在研究中可以达成多种目的，比如对基因进行切割、修饰、沉默、敲除、调整表达等操作。CRISPR/Cas已经成为基因编辑和研究的有力工具，具有十分光明而广泛的应用前景。
作为CRISPR/Cas系统中不可或缺的sgRNA，其合成方法非常重要，如何以较低的成本获得可用的sgRNA成为科研工作者的关注目标。化学合成RNA法时间周期为7～14天，而且费用很高，不适合sgRNA合成。构建质粒载体在体内转录RNA的方法需要繁琐费时的载体构建工作，而且不够灵活，每个载体只能对应少量特定的RNA。
而目前传统的sgRNA体外转录法有很多繁琐的步骤，通常是1、PCR扩增模板DNA，2、胶回收PCR产物，3、将PCR产物作为模板转录sgRNA。其操作费时费力，整个流程需要5～6小时。其间需要使用PCR仪、电泳槽、离心机等多种仪器，涉及的试剂有PCR扩增酶、胶回收试剂盒、T 7RNA酶等，因此平均费用不低。
因此本领域迫切地需要开放一种简单、方便、省时、高效又节省的sgRNA体外合成方法。
发明内容
本发明提供了一种简单、方便、省时、高效又节省的sgRNA体外合成方法。
本发明第一方面提供了一种sgRNA合成体系，包括：
(a)核酸构建物，所述核酸构建物具有5’-3’的式I所示的结构：
Y1-L1-Y2-L2-Y3-Y4   (I)
其中，
Y1为RNA聚合酶启动区；
L1为无或连接序列；
Y2为靶DNA序列；
L2为无或连接序列；
Y3为下游引物结合区；
Y4为无或核苷酸序列；
并且，各“-”独立地为键或核苷酸连接序列；
(b)下游引物；
(c)DNA聚合酶；和
(d)RNA聚合酶。
在另一优选例中，所述RNA聚合酶选自下组：T7RNA聚合酶、Sp6RNA聚合酶、U6RNA聚合酶、T3RNA聚合酶、或其组合。
在另一优选例中，所述RNA聚合酶启动区为T7RNA聚合酶启动区。
在另一优选例中，所述元件Y1的序列结构为：N X-TAATACGACTCACTATA(SEQ ID NO.:1的第2-18位)-G Y，其中N为A、T、C或G，X为1-6的整数，Y为0-2的整数。
在另一优选例中，所述元件Y1的序列如SEQ ID NO.:1所示。
在另一优选例中，所述元件Y1的序列如SEQ ID NO.:2所示。
在另一优选例中，所述元件Y1的序列如SEQ ID NO.:3所示。
在另一优选例中，所述元件Y1的长度为17-40bp，较佳地，18-25bp。
在另一优选例中，所述元件Y2的长度为0-100bp，较佳地，0-90bp。
在另一优选例中，所述元件Y3的长度为5-30bp，较佳地，10-20bp。
在另一优选例中，所述元件Y4的长度为5-30bp，较佳地8-20bp。
在另一优选例中，所述连接序列的长度为1-30nt。
在另一优选例中，所述L2和/或Y3中的一部分或全部可作为barcode序列使用。
在另一优选例中，所述L2和/或Y3含有barcode序列。
在另一优选例中，所述L2为barcode序列。
在另一优选例中，所述Y3为通用引物结合区。
在另一优选例中，所述下游引物包括特异性引物、通用引物、barcode引物。
在另一优选例中，所述下游引物被用作通用引物或barcode引物。
在另一优选例中，所述组分(d)与组分(a)中的RNA聚合酶启动区相对应。
在另一优选例中，所述sgRNA合成体系还包括选自下组的一种或多种组分：
(e)DTT；
(f)亚精胺；
(g)甘油；
(h)无RNA酶的水(RNase Free water)；
(i)Triton-X100；
(j)RNA酶抑制剂；
(k)硫酸铵；
(l)吐温20
(m)用于合成RNA的底物；
(n)用于合成DNA的底物；
(o)镁离子；
(p)缓冲剂。
在另一优选例中，所述的合成RNA的底物包括：核苷单磷酸、核苷三磷酸、或其组合。
在另一优选例中，所述合成DNA的底物包括：脱氧核苷单磷酸、脱氧核苷三磷酸、或其组合。
在另一优选例中，所述DNA聚合酶选自下组：Klenow聚合酶、Taq酶、Pfu酶、KOF酶、Bst酶、Phi29酶、或其组合。
在另一优选例中，所述RNA聚合酶选自下组：T7RNA聚合酶、Sp6RNA聚合酶、U6RNA聚合酶、T3RNA聚合酶、或其组合。
在另一优选例中，所述镁离子来源于镁离子源，所述镁离子源选自下组：氯化镁、醋酸镁、谷氨酸镁、磷酸镁、或其组合。
在另一优选例中，所述缓冲剂选自下组：Tris-HCl、4-羟乙基哌嗪乙磺酸、三羟甲基氨基甲烷、磷酸盐缓冲液、柠檬酸缓冲液、或其组合。
在另一优选例中，所述sgRNA合成体系中，组分(a)的质量浓度(wt％)为0.0008％-0.006％，较佳地，0.001％-0.005％，更佳地，0.0013％-0.00438％，以所述sgRNA合成体系的总重计。
在另一优选例中，所述sgRNA合成体系中，组分(a)的摩尔浓度(mol/L)为0.3umol/L-2umol/L，较佳地，0.5umol/L-1.5umol/L，更佳地，0.8umol/L-1.2umol/L，以所述sgRNA合成体系的总体积计。
在另一优选例中，所述sgRNA合成体系中，组分(b)的质量浓度(wt％)为0.00024％-0.0011％，较佳地，0.0003％-0.00093％，更佳地，0.00039％-0.00081％，以所述sgRNA合成体系的总重计。
在另一优选例中，所述sgRNA合成体系中，组分(c)的酶活(U/uL)为0.05U/uL-0.4U/uL，较佳地，0.08U/uL-0.25U/uL，更佳地，0.1U/uL-0.2U/uL，以所述sgRNA合成体系的总体积计。
在另一优选例中，所述sgRNA合成体系中，组分(d)的酶活(U/uL)为1U/uL-6U/uL，较佳地，2U/uL-5U/uL，更佳地，3U/uL-4U/uL，以所述sgRNA合成体系的总体积计。
在另一优选例中，所述sgRNA合成体系中，组分(e)的摩尔浓度(mol/L)为0.1mmol/L-3mmol/L，较佳地，0.3mmol/L-2mmol/L，更佳地，0.5mmol/L-1.5mmol/L，以所述sgRNA合成体系的总体积计。
在另一优选例中，所述sgRNA合成体系中，组分(f)的摩尔浓度(mol/L)为0.8mmol/L-4mmol/L，较佳地，1.2mmol/L-3mmol/L，更佳地，1.5mmol/L-2.5mmol/L，以所述sgRNA合成体系的总重计。
在另一优选例中，所述sgRNA合成体系中，组分(g)的体积浓度(v/v)为15％-35％，较佳地，18％-32％，更佳地，20％-30％，以所述sgRNA合成体系的总体积计。
在另一优选例中，所述sgRNA合成体系中，组分(j)的体积浓度(v/v)为5％-15％， 较佳地，6％-13％，更佳地，8％-11％，以所述sgRNA合成体系的总体积计。
在另一优选例中，所述sgRNA合成体系中，组分(l)的体积浓度(v/v)为0.2％-0.7％，较佳地，0.3％-0.6％，更佳地，0.4％-0.6％，以所述sgRNA合成体系的总重计。
在另一优选例中，所述sgRNA合成体系中，组分(m)的摩尔浓度(mol/L)为1mmol/L-1.5mmol/L，较佳地，1.1mmol/L-1.4mmol/L，更佳地，1.2mmol/L-1.3mmol/L，以所述sgRNA合成体系的总重计。
在另一优选例中，所述sgRNA合成体系中，组分(n)的摩尔浓度(mol/L)为0.3mmol/L-0.7mmol/L，较佳地，0.35mmol/L-0.6mmol/L，更佳地，0.4mmol/L-0.5mmol/L，以所述sgRNA合成体系的总重计。
在另一优选例中，所述组分(m)/组分(n)为10:1-4:1，较佳地，8:1-3:1，更佳地，5:1-2:1。
在另一优选例中，所述sgRNA合成体系中，组分(o)的摩尔浓度(mol/L)为3.5mmol/L-7mmol/L，较佳地，4mmol/L-6.5mmol/L，更佳地，5mmol/L-6mmol/L，以所述sgRNA合成体系的总重计。
在另一优选例中，所述的sgRNA合成体系具有以下性能：
在合成体系(5ul－200ul，较佳地，10-100ul)里，sgRNA合成总量≥1ug(1-10ug，较佳地，1-5ug)。
本发明第二方面提供了一种体外合成sgRNA的方法，包括步骤：
(i)提供本发明第一方面所述的sgRNA合成体系；
(ii)在适合的条件下，孵育步骤(i)的合成体系一段时间T1，从而合成所述的sgRNA。
另一优选例中，所述的方法还包括：(iii)任选地从所述sgRNA合成体系中，分离或检测所述的sgRNA。
在另一优选例中，所述步骤(ii)中，反应温度为25℃-42℃，较佳地，35℃-40℃。
在另一优选例中，所述步骤(ii)中，反应时间T1为0.5h-8h，较佳地，0.5-3h，较佳地，0.8h-1h。
本发明第三方面提供了一种用于sgRNA合成的试剂盒，包括：
(k1)第一容器，以及位于第一容器内的核酸构建物，所述核酸构建物具有5’-3’的式I所示的结构：
Y1-L1-Y2-L2-Y3-Y4   (I)
其中，
Y1为RNA聚合酶启动区；
L1为无或连接序列；
Y2为靶DNA序列；
L2为无或连接序列；
Y3为下游引物结合区；
Y4为无或核苷酸序列；
并且，各“-”独立地为键或核苷酸连接序列；
(k2)第二容器，以及位于第二容器内的下游引物；和
(kt)标签或说明书。
在另一优选例中，所述的第一容器、第二容器是同一容器或不同容器。
在另一优选例中，所述试剂盒还包括任选的选自下组的一个或多个容器：
(k3)第三容器，以及位于第三容器内的DNA聚合酶；
(k4)第四容器，以及位于第四容器内的RNA聚合酶；
(k5)第五容器，以及位于第五容器内的用于合成RNA的底物；
(k6)第六容器，以及位于第六容器内的用于合成DNA的底物；
(k7)第七容器，以及位于第七容器内的镁离子；
(k8)第八容器，以及位于第八容器内的缓冲剂。
本发明第四方面提供了一种核酸构建物，所述核酸构建物具有5’-3’的式I所示的结构：
Y1-L1-Y2-L2-Y3-Y4   (I)
其中，
Y1为RNA聚合酶启动区；
L1为无或连接序列；
Y2为靶DNA序列；
L2为无或连接序列；
Y3为下游引物结合区；
Y4为无或核苷酸序列；
并且，各“-”独立地为键或核苷酸连接序列。
在另一优选例中，所述RNA聚合酶选自下组：T7RNA聚合酶、Sp6RNA聚合酶、U6RNA聚合酶、T3RNA聚合酶、或其组合。
在另一优选例中，所述RNA聚合酶启动区为T7RNA聚合酶启动区。
在另一优选例中，所述元件Y1的序列结构为：N X-TAATACGACTCACTATA(SEQ ID NO.:1的第2-18位)-G Y，其中N为A、T、C或G，X为1-6的整数，Y为0-2的整数。
在另一优选例中，所述元件Y1的序列如SEQ ID NO.:1所示。
在另一优选例中，所述元件Y1的序列如SEQ ID NO.:2所示。
在另一优选例中，所述元件Y1的序列如SEQ ID NO.:3所示。
在另一优选例中，所述元件Y1的长度为17-40bp，较佳地，18-25bp。
在另一优选例中，所述元件Y2的长度为0-100bp，较佳地，0-90bp。
在另一优选例中，所述元件Y3的长度为5-30bp，较佳地，10-20bp。
在另一优选例中，所述元件Y4的长度为5-30bp，较佳地8-20bp。
在另一优选例中，所述连接序列的长度为1-30nt。
在另一优选例中，所述L2和/或Y3中的一部分或全部可作为barcode序列使用。
在另一优选例中，所述L2和/或Y3含有barcode序列。
在另一优选例中，所述L2为barcode序列。
在另一优选例中，所述Y3为通用引物结合区。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了A、B方案体外转录合成sgRNA电泳图。A、B分别代表方案A、B实验体系；泳道1为Marker；泳道2-5分别为0.5h、1h、1.5h、2h反应时长。
图2显示了C、D方案体外转录合成sgRNA电泳图。C、D分别代表方案C、D实验体系；泳道1为Marker；泳道2-5分别为0.5h、1h、1.5h、2h反应时长。
图3显示了：E方案体外转录合成sgRNA电泳图。泳道5为Marker；泳道1-4分别为0.5h、1h、1.5h、2h反应时长。
图4显示了F方案与C方案sgRNA体外转录对比电泳图。泳道2为DNA Marker 2000；泳道1为方案F；泳道3为方案C。
图5显示了阳性代表CRISPR/Cas切割体系中加入阳性对照sgRNA；MK为DNA Ladder 2000；A1、A2、A3、A4分别代表CRISPR/Cas切割体系中加入A方案中四个反应时长所得的sgRNA，即0.5h，1h，1.5h，2h；B1-4，C1-4，D1-4以此类推为加入B、C、D方案四个反应时长所得的sgRNA；F1、F2为F方案sgRNA扩增时长分别为3h、2h。
具体实施方式
经过广泛而深入的研究，通过大量筛选和摸索，首次意外地发现了一种简单、方便、省时、高效又节省的sgRNA体外合成方法。具体地，本发明的sgRNA合成体系可显著提高sgRNA的合成产量，产量≥1ug(1-10ug，较佳地，1-5ug)，并且可以将整个流程时间大幅缩短(如降低到1小时以内)，步骤简便，平均费用也大大下降(下降约50％)。在此基础上，本发明人完成了本发明。
本发明的构建物
本发明提供了一种核酸构建物，其是通过将RNA聚合酶启动区(如T7RNA聚合酶启动区)、靶DNA序列、下游引物结合区(如特异性引物、通用引物、barcode引物或可被用作通用引物、barcode引物的下游引物)以及任选的额外的核苷酸序列通过连接序列连接起来，从而可简单、方便、省时、高效的体外合成sgRNA，并且成本很低。本发明的构建物如本发明第一方面所述。
本发明的构建物中所用的各种元件都是本领域中已知的，因此本领域技术人员可以用常规方法，如PCR方法、全人工化学合成法、酶切方法获得相应的元件，然后通过熟知的DNA连接技术连接在一起，就形成了本发明的构建物。
在本发明中，RNA聚合酶启动区的序列结构为：N X-TAATACGACTCACTATA (SEQ ID NO.:1的第2-18位)-G Y，其中N为A、T、C或G，X为1-6的整数，Y为0-2的整数。
在一优选实施方式中，RNA聚合酶启动区为T7聚合酶启动区，序列如SEQ ID NO.:1所示。
在一优选实施方式中，RNA聚合酶启动区为SP6聚合酶启动区，序列如SEQ ID NO.:2所示。
在一优选实施方式中，RNA聚合酶启动区为T3聚合酶启动区，序列如SEQ ID NO.:3所示。
sgRNA合成体系
本发明提供了一种体外的sgRNA合成体系，包括：
(a)本发明第一方面所述的核酸构建物；
(b)下游引物；
(c)DNA聚合酶；和
(d)RNA聚合酶。
在一优选实施方式中，所述体外的sgRNA合成体系还包括：
(e)DTT；
(f)亚精胺；
(g)甘油；
(h)无RNA酶的水(RNase Free water)；
(i)Triton-X100；
(j)RNA酶抑制剂；
(k)硫酸铵；
(l)吐温20
(m)用于合成RNA的底物；
(n)用于合成DNA的底物；
(o)镁离子；
(p)缓冲剂。
在本发明中，所述下游引物没有特别限制，可以包括通用引物或barcode引 物，或可被用作通用引物或barcode引物。通常，下游引物的浓度为1uM。
在本发明中，RNA聚合酶没有特别限制，可以选自一种或多种RNA聚合酶，典型的RNA聚合酶为T7RNA聚合酶。通常，RNA聚合酶的浓度为3.5U/ul。
在本发明中，DNA聚合酶没有特别限制，可以选自一种或多种DNA聚合酶，包括(但并不限于)：Klenow聚合酶、Taq酶、Pfu酶、KOF酶、Bst酶、和/或Phi29酶。一种典型的DNA聚合酶为Taq酶。通常，DNA聚合酶的浓度为0.15U/ul。
在本发明中，所述sgRNA合成体系中的核苷三磷酸混合物为腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸。在本发明中，各种单核苷酸的浓度没有特别限制，通常每种单核苷酸的浓度为0.1mM-0.5mM，较佳地为0.25mM-0.35mM。
在本发明中，所述sgRNA合成体系中的脱氧核苷三磷酸混合物为腺嘌呤脱氧核苷三磷酸、鸟嘌呤脱氧核苷三磷酸、胞嘧啶脱氧核苷三磷酸和胸腺嘧啶脱氧核苷三磷酸。在本发明中，各种单脱氧核苷酸的浓度没有特别限制，通常每种单脱氧核苷酸的浓度为0.05mM-0.15mM，较佳地为0.1mM-0.13mM。
在本发明中，组分(e)-(p)的浓度没有特别限制，合成sgRNA可用常用浓度的组分(e)-(p)。
在一特别优选的实施方式中，所述sgRNA合成体系包括：
Tris-HCl 40mM
MgCl 2 5-6mM
DTT 0.8-1mM
亚精胺 1.8-2.5mM
A组分 0.5-1μM
B组分 0.5-1 μM
dNTPs 0.5-1mM
NTPs 1.25-1.5mM
T7 RNA聚合酶 3.5-5U/ul
Taq DNA聚合酶 0.15-0.2U/ul。
试剂盒
本发明提供了用于sgRNA合成的试剂盒，包括：
(k1)第一容器，以及位于第一容器内的本发明第一方面所述的核酸构建物；
(k2)第二容器，以及位于第二容器内的下游引物；和
(kt)标签或说明书。
在一优选实施方式中，所述的第一容器、第二容器是同一容器或不同容器。
一种特别优选的体外sgRNA合成的试剂盒包含一个体外sgRNA合成体系，该合成体系包括：Tris-HCl 40mM，MgCl 2 6mM，DTT 1mM，亚精胺2mM，A组分1uM，B组分1uM，dNTPs 0.5mM，NTPs 1.25mM，T7RNA聚合酶3.5U/ul,Taq DNA聚合酶0.15U/ul。
本发明的主要优点包括：
(1)本发明首次开发一种简单、方便、省时、高效又节省的sgRNA体外合成方法。
(2)本发明的方法可以将整个流程时间大幅降低(如降低到1小时以内)，步骤简便，其平均费用也大大下降(降了约50％)。
(3)本发明的方法去除了传统RNA体外转录法的PCR扩增及胶回收步骤，将整个流程的时长从4～6小时降低到1小时。本发明还减少了所需添加的试剂，令整个操作更加简便。
(4)本发明的方法在省去了PCR扩增后，转录生成的RNA量远超传统转录法，在减少了总时长的情况下，一步法的产量还超过了传统的RNA体外转录法，产量≥1ug(1-10ug，较佳地，1-5ug)。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，则本发明实施例中所用的材料和试剂均为市售产品。
通用方法
在本发明中，sgRNA体外一步转录合成反应用的反应缓冲液，其具体成分为：Tris-HCl，MgCl 2，DTT，亚精胺，sgRNA-R，dNTPs，NTP。
优选的，Buffer具体为：
反应缓冲液 终浓度
Tris-HCl 40mM
MgCl 2 6mM
DTT 1mM
亚精胺 2mM
sgRNA-R 1uM
dNTPs 1.25mM
NTP 0.5mM
在本发明中，还提供一种sgRNA体外一步转录合成反应用的Enzyme Mix，Mix是两种酶的混合物，一种是DNA聚合酶(Klenow聚合酶，Taq酶，Pfu酶，KOF酶，Bst酶，Phi29酶中的一种)，第二种是RNA聚合酶(T 7酶，Sp6酶，U6酶中的一种)。
优选的，Enzyme Mix为Taq酶与T 7RNA聚合酶的组合。
本发明的实施步骤为：
1.sgRNA模板引物设计
1.sgRNA的正向(F)引物设计
选取Target DNA序列PAM(NGG)的5’端20bp进行正向(F)引物设计，引物结构包含T7promoter(20bp)、Target DNA片段(20bp)和实际与反向引物结合的片段(10～25bp)。
引物设计示意图如下所示：
Figure PCTCN2019080485-appb-000001
例如，假设Target DNA片段为
5’-TCCCCGCTGCAGCATAGTGAGCCCAGAANGGCATT-3’(SEQ ID NO.：4)，则引物设计的示意图如下所示：
Figure PCTCN2019080485-appb-000002
II.sgRNA体外转录体系
按照引物设计要求设计并合成正向引物，按如下体系配制，并在37℃反应1h：
转录体系 加量(μL)
反应缓冲液 10
正向引物F(10μM) 2
Enzyme Mix 2
RNase Free Water To 40
III.sgRNA纯化
3.1模板DNA的去除：
向转录产物中加1μl DNase I，37℃反应10min，以去除DNA模板，将反应产物放入75℃反应10min，得到的sgRNA置于-20℃保存。
3.2sgRNA纯化(可选)：
1.转录体系20μl用RNase Free Water补至300μl，加入等体积酚·氯仿·异戊醇(25∶24∶1)，充分混合后，12000rpm，4℃离心15min。
2.取上清，加入300μl氯仿，充分混匀后，12000rpm，4℃离心5min。
3.取上清约200μl，为提高回收量，下层可再加入100μl RNase Free Water，充分混合后，4℃离心5min，取上清。
4.将两次取的上清合并，总体积约300～350μl，加入2倍体积的异丙醇和1/10体积的用RNase Free Water配制的3M NaAc，-80℃沉淀过夜。
5.离心取沉淀，加入1ml RNase Free Water配制的75％乙醇，洗涤两次。
6.用20μl RNase Free Water溶解RNA，电泳检测质量并进行浓度测定。
实施例1 设计A、B、C、D、E五个体外转录体系，另设计F为传统体外转录体系，比较方案优劣。
设计sgRNA正向引物(sgRNA-F)：
5’-TTAATACGACTCACTATAGGGCAGCATAGTGAGCCCAGAAGTTTTAGAGCTAGAAATAGCA-3’(SEQ ID NO.：5)
设计sgRNA反向引物(sgRNA-R)：
5’-TGCTATTTCTAGCTCTAAAAC-3’(SEQ ID NO.：6)
将sgRNA-F与sgRNA-R溶于RNase Free Water，配制成5uM浓度。
1、A与B方案实验体系，将除了引物以外的所有组分混合到一起，配制成反应缓冲液1，其各组分如下：
Figure PCTCN2019080485-appb-000003
A、B方案按照如下配制20μL反应体系：
Figure PCTCN2019080485-appb-000004
使用A、B方案分别各设置4次重复，置于PCR仪，37℃条件下反应，4次重复分别反应时长为0.5h、1h、1.5h、2h。反应结束后加入1μl DNase Ⅰ，37℃反应10min，以去除DNA模板，将产物进行电泳，其结果如图1。
结果表明，随着反应时间从0.5h到2h，产量提高；A方案的产量高于B方案，其中，A方案的产量为1.2ug(2h)，B方案的产量为1ug(2h)。
2、C与D方案实验体系，将除了正向引物和两种酶之外的其他组分混合，配制成反应缓冲液2，其各组分如下：
反应缓冲液2 C方案(μL) D方案(μL)
Tris-HCl 80 80
MgCl 2 24 24
DTT 2 2
亚精胺 40 40
sgRNA-R 200 200
dNTPs(10mM) 100 100
NTP(25mM) 100 200
RNase Free Water 454 354
总体积 1000 1000
将Taq酶和T7RNA酶混合在一起，配制成Enzyme Mix：
Figure PCTCN2019080485-appb-000005
C、D方案按照如下配制20μL反应体系：
反应总体积           20μL
反应缓冲液2          10μL
sgRNA-F              2μL
酶混合物
(Enzyme Mix)         2μL
RNase Free Water     6μL
使用C、D方案分别各设置4次重复，置于PCR仪，37℃条件下反应，4次重复分别反应时长为0.5h、1h、1.5h、2h。反应结束后加入1 μl DNase I，37℃反应10min，以去除DNA模板，将产物进行电泳，其结果如图2。
结果表明，随着反应时间从0.5h到2h，产量提高；C方案的产量远远高于D方案，其中C方案的产量为2ug(2h)，D方案的产量为400ng(2h)。
3、E方案，分别配制Transcription Mix：
转录混合物(Transcription Mix) 体积(μL)
Tris-HCl 80
MgCl 2 32
DTT 10
亚精胺 40
NTP(25mM) 80
Triton-X100 10
甘油(原液) 150
RNase抑制剂 100
T7 RNA聚合酶 150
RNase Free Water 388
总体积 1000
和模板扩增混合物(Template Amplification Mix)：
模板扩增混合物(Template Amplification Mix) 体积(μL)
Tris-HCl 375
(NH 4) 2SO 4 100
MgCl 2 20
Tween 20 50
DTT 2.5
甘油(原液) 250
HotStart Taq DNA聚合酶 80
RNase Free Water 122.5
总体积 1000
E方案按照如下配制20μL反应体系：
Figure PCTCN2019080485-appb-000006
Figure PCTCN2019080485-appb-000007
使用E方案做4次重复，置于PCR仪，37℃条件下反应，4次重复反应时长分别为0.5h、1h、1.5h、2h。反应结束后加入1μl DNase I，37℃反应10min，以去除DNA模板，将产物进行电泳，其结果如图3。
结果表明，随着反应时间从0.5h到2h，产量提高，E方案的产量很低，E方案的产量为200ng(2h)。
4、F方案，分别配制Transcription Mix：
Transcription Mix 体积(μL)
Tris-HCl 80
MgCl 2 32
DTT 10
亚精胺 40
NTP(25mM) 80
Triton-X100 10
甘油(原液) 150
RNase抑制剂 100
T7 RNA聚合酶 150
RNase Free Water 388
总体积 1000
和Template Amplification Mix：
Figure PCTCN2019080485-appb-000008
将正向引物、反向引物和Template Amplification Mix配制成如下20μL PCR反应体系：
Figure PCTCN2019080485-appb-000009
将反应体系放入PCR仪，按照如下程序进行PCR扩增：
Figure PCTCN2019080485-appb-000010
将PCR扩增产物配制如下20μL反应体系：
PCR产物              10μL
Transcription Mix    10μL
37℃，反应2小时，其sgRNA合成结果与C方案比较如图4。
结果表明，方案C效果最佳，其合成的sgRNA产量最高，可达1ug-2ug。并且意外的，其产量远超传统sgRNA合成法(传统合成法的sgRNA产量约为 100ng-200ng)，提高了5-10倍。
实施例2 检测一步法体外转录合成的sgRNA在CRISPR/Cas系统内的活性效果
CRISPR/Cas9体外切割反应体系：
Figure PCTCN2019080485-appb-000011
配制19个体系，1个体系中加入阳性对照sgRNA，其余的分别加入体外转录所得的sgRNA，37℃反应1h，70℃反应10min，10℃反应10min。
反应结束后电泳结果如图5所示。
结果表明，当反应体系中的NTP/dNTP为10∶1-4∶1，较佳地，8∶1-3∶1，更佳地，5∶1-2∶1时，本发明合成的sgRNA具有引导Cas9切割特异位点的活性。
并且，除了D方案0.5h转录时长所得的sgRNA因为量太少，无法引导Cas9蛋白切割DNA之外，其他所有DNA均被sgRNA引导的Cas9蛋白切割。这进一步表明通过本发明一步体外转录法合成的sgRNA具有引导Cas9切割特异位点的活性。
C方案与D方案的对比：
反应缓冲液2 C方案(μL) D方案(μL)
Tris-HCl 80 80
MgCl 2 24 24
DTT 2 2
亚精胺 40 40
sgRNA-R 200 200
dNTPs(10mM) 100 100
NTPs(25mM) 100 200
RNase Free Water 454 354
总体积 1000 1000
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (16)

  1. 一种sgRNA合成体系，其特征在于，包括：
    (a)核酸构建物，所述核酸构建物具有5’-3’的式I所示的结构：
    Y1-L1-Y2-L2-Y3-Y4  (I)
    其中，
    Y1为RNA聚合酶启动区；
    L1为无或连接序列；
    Y2为靶DNA序列；
    L2为无或连接序列；
    Y3为下游引物结合区；
    Y4为无或核苷酸序列；
    并且，各“-”独立地为键或核苷酸连接序列；
    (b)下游引物；
    (c)DNA聚合酶；和
    (d)RNA聚合酶。
  2. 如权利要求1所述的sgRNA合成体系，其特征在于，所述RNA聚合酶选自下组：T7RNA聚合酶、Sp6RNA聚合酶、U6RNA聚合酶、T3RNA聚合酶、或其组合。
  3. 如权利要求1所述的sgRNA合成体系，其特征在于，所述L2和/或Y3中的一部分或全部可作为barcode序列使用。
  4. 如权利要求1所述的sgRNA合成体系，其特征在于，所述L2为barcode序列。
  5. 如权利要求1所述的sgRNA合成体系，其特征在于，所述Y3为通用引物结合区。
  6. 如权利要求1所述的sgRNA合成体系，其特征在于，所述元件Y1的序列结构为：N X-TAATACGACTCACTATA(SEQ ID NO.:1的第2-18位)-G Y，其中N为A、T、C或G，X为1-6的整数，Y为0-2的整数。
  7. 如权利要求1所述的sgRNA合成体系，其特征在于，所述sgRNA合成体系还包括选自下组的一种或多种组分：
    (e)DTT；
    (f)亚精胺；
    (g)甘油；
    (h)无RNA酶的水(RNase Free water)；
    (i)Triton-X100；
    (j)RNA酶抑制剂；
    (k)硫酸铵；
    (l)吐温20
    (m)用于合成RNA的底物；
    (n)用于合成DNA的底物；
    (o)镁离子；
    (p)缓冲剂。
  8. 一种体外合成sgRNA的方法，其特征在于，包括步骤：
    (i)提供权利要求1所述的sgRNA合成体系；
    (ii)在适合的条件下，孵育步骤(i)的合成体系一段时间T1，从而合成所述的sgRNA。
  9. 如权利要求8所述的方法，其特征在于，所述的方法还包括：(iii)任选地从所述sgRNA合成体系中，分离或检测所述的sgRNA。
  10. 一种用于sgRNA合成的试剂盒，其特征在于，包括：
    (k1)第一容器，以及位于第一容器内的核酸构建物，所述核酸构建物具有5’-3’的式I所示的结构：
    Y1-L1-Y2-L2-Y3-Y4  (I)
    其中，
    Y1为RNA聚合酶启动区；
    L1为无或连接序列；
    Y2为靶DNA序列；
    L2为无或连接序列；
    Y3为下游引物结合区；
    Y4为无或核苷酸序列；
    并且，各“-”独立地为键或核苷酸连接序列；
    (k2)第二容器，以及位于第二容器内的下游引物；和
    (kt)标签或说明书。
  11. 一种核酸构建物，其特征在于，所述核酸构建物具有5’-3’的式I所示的结构：
    Y1-L1-Y2-L2-Y3-Y4  (I)
    其中，
    Y1为RNA聚合酶启动区；
    L1为无或连接序列；
    Y2为靶DNA序列；
    L2为无或连接序列；
    Y3为下游引物结合区；
    Y4为无或核苷酸序列；
    并且，各“-”独立地为键或核苷酸连接序列。
  12. 如权利要求11所述的核酸构建物，其特征在于，所述RNA聚合酶启动区为T7RNA聚合酶启动区。
  13. 如权利要求11所述的核酸构建物，其特征在于，所述元件Y1的序列结构为：N X-TAATACGACTCACTATA(SEQ ID NO.:1的第2-18位)-G Y，其中N为A、T、C或G，X为1-6的整数，Y为0-2的整数。
  14. 如权利要求11所述的核酸构建物，其特征在于，所述元件Y1的序列如SEQ ID NO.:1所示。
  15. 如权利要求11所述的核酸构建物，其特征在于，所述元件Y1的序列如SEQ ID NO.:2所示。
  16. 如权利要求11所述的核酸构建物，其特征在于，所述元件Y1的序列如SEQ ID NO.:3所示。
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