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In-vitro synthesis method of sgRNA and kit adopting same

A technology of RNA polymerase and synthesis system, applied in DNA/RNA fragment, DNA preparation, recombinant DNA technology, etc., can solve the problem of time-consuming and laborious operation

Active Publication Date: 2018-07-27
NOVOPROTEIN SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its operation is time-consuming and laborious, and the whole process takes 5-6 hours

Method used

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  • In-vitro synthesis method of sgRNA and kit adopting same
  • In-vitro synthesis method of sgRNA and kit adopting same
  • In-vitro synthesis method of sgRNA and kit adopting same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0209] Example 1 Five in vitro transcription systems 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 schemes were compared.

[0210] Design sgRNA forward primer (sgRNA-F):

[0211] 5'-TTAATACGACTCACTATAGGGCAGCATAGTGAGCCCAGAAGTTTTTAGAGCTAGAAATAGCA-3' (SEQ ID NO.: 5)

[0212] Design sgRNA reverse primer (sgRNA-R):

[0213] 5'-TGCTATTTCTAGCTCTAAAAC-3' (SEQ ID NO.: 6)

[0214] Dissolve sgRNA-F and sgRNA-R in RNase Free Water to prepare a concentration of 5uM.

[0215] 1. For the experimental system of scheme A and B, mix all components except primers together to prepare reaction buffer 1, and its components are as follows:

[0216]

[0217] Prepare a 20 μL reaction system for A and B schemes as follows:

[0218] Total reaction volume 20 μL

[0219] Reaction buffer 1 10 μL

[0220] sgRNA-F 2 μL

[0221] sgRNA-R 2 μL

[0222] RNase Free Water 6μL

[0223] Set up 4 repetitions ...

Embodiment 2

[0271] Example 2 Detection of the activity effect of the sgRNA synthesized by one-step in vitro transcription in the CRISPR / Cas system

[0272]

[0273] Prepare 19 systems, add positive control sgRNA to 1 system, and add sgRNA obtained from in vitro transcription to the rest, react at 37°C for 1h, 70°C for 10min, and 10°C for 10min.

[0274] After the reaction, the electrophoresis results were as follows: Figure 5 shown.

[0275] The results show that when the NTP / dNTP in the reaction system is 10:1-4:1, preferably 8:1-3:1, more preferably 5:1-2:1, the sgRNA synthesized by the present invention It has the activity of guiding Cas9 to cut specific sites.

[0276] Moreover, except that the amount of sgRNA obtained by the 0.5h transcription time of the D scheme was too small to guide the Cas9 protein to cut DNA, all other DNA was cut by the sgRNA-guided Cas9 protein. This further indicates that the sgRNA synthesized by the one-step in vitro transcription method of the prese...

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Abstract

The invention provides an in-vitro synthesis method of sgRNA and a kit adopting the same, particularly, the invention provides a nucleic acid construction object Y1-L1-Y2-L2-Y3-Y4 (I), the structure of the nucleic acid construction object is shown in a 5'-3'formula I, wherein Y1 is an RNA polymerase promotor; L1 is none or a connecting sequence; Y2 is a target DNA sequence; L2 is none or a connecting sequence; Y3 is a downstream primer combination region; Y4 is none or a nucleotide sequence, and - is independently a bond or a nucleotide sequence. The sgRNA synthesis system obviously improves the synthesis yield of the sgRNA, the whole flow time is greatly shortened, the steps are convenient, and the average cost is also greatly lowered.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an in vitro synthesis method of sgRNA and a kit thereof. Background technique [0002] CRISPR (clustered regularly interspaced short palin-dromic repeats) / Cas (CRISPR-associated) system is a unique acquired immune system in some bacteria and archaea. This system is guided by a specific sequence of RNA to specifically cut and degrade exogenous DNA. . The CRISPR / Cas system can be divided into three types, among which the type II CRISPR / Cas system has been transformed into a tool for targeted genome editing due to its simple composition. By artificially designing and transcribing RNA in vitro, sgRNA (single guide RNA) with a guiding effect can be synthesized, and the sgRNA guides the Cas protein to specifically cut the target DNA sequence. Through the modification of the Cas protein, under the guidance of sgRNA, the CRISPR / Cas system can achieve a variety of purposes in research, such...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/10C12N15/113C12N9/22
CPCC12N9/22C12N15/10C12N15/113C12N15/63C12N2310/10C12N2310/20C12P19/34C12N2330/50C12N15/111C12N15/11C12N2800/80
Inventor 朱化星张清仪石加加赵曼曼何翼
Owner NOVOPROTEIN SCI INC