Synthetic single guide RNA for Cas9-mediated gene editing

A single-guide and composition technology, applied in the field of gene editing, can solve the problems of low yield and the impossibility of practical application of chemical synthesis of single-guide RNA molecules

Inactive Publication Date: 2018-02-16
达尔马科恩有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, due to their size (~116nt) and low yield, the chemical synthesis of single-guide RNA molecules has not been practically possible

Method used

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  • Synthetic single guide RNA for Cas9-mediated gene editing
  • Synthetic single guide RNA for Cas9-mediated gene editing
  • Synthetic single guide RNA for Cas9-mediated gene editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1. Preparation of 3'-azidoadenosine polystyrene support ( figure 1 )

[0078] 1.

[0079] N 6 -Isobutyryl-2'-O-[2-(2-hydroxyethyl)methylcarbamate]-3',5'-O-(tetraisopropyl-di Siloxane-1,3-diyl)adenosine (2):

[0080] To a solution of compound 1 (10.0 g, 17.2 mmol) in 170 mL of dichloromethane (DCM) was added CDI (1,1'-carbonyldiimidazole) (2.9 g, 18.1 mmol). After stirring for 18 hours, 2-(methylamino)ethanol (5.2 g, 68.8 mmol) was added. After 1.5 hours the reaction was stopped and evaporated to dryness. The crude material was purified on a Biotage Isolera using a 100 g Ultra column with an ethyl acetate:MeOH gradient (0→10%) to afford 2 (10.8 g, 93%) as a white foam. Compound 2 was analyzed by RP-HPLC: 10.54 min, 99.4%. 1 H NMR (CDCl 3 , 300MHz) δ8.65(s, 1H), 8.63(s, 1H), 8.10(s, 1H), 6.04(d, J=8.8Hz, 1H), 5.64(d, J=5.3Hz, 1H), 5.15 (m, 1H), 4.16-3.98 (m, 4H), 3.76 (m, 2H), 3.56-3.15 (m, 3H), 3.05 and 2.96 (s, 3H each), 2.86 (s, 1H), 2.59 (m, 1H), ...

Embodiment 2

[0091] Embodiment 2. prepare 5'-hexyne phosphoramidite (8) ( figure 2 )

[0092] Compound 7 (hex-5-yn-1-ol, 1.4 mL) was dissolved in 10 mL of DCM in a flask, and N,N-diisopropylamine (1.82 mL) was added to the solution. In another flask under anhydrous conditions, dilute the phosphitylation reagent bis-(N,N-diisopropylamino)-cyanoethylphosphine (1.5 eq / eq 7), MeCN containing 0.45M 1H-tetrazole (0.5 eq tetrazole / eq 7) was added and shaken for 5 min. Next, the solution of the activated phosphitylation reagent was added to the well-stirred solution of compound 7 at room temperature, and stirred at room temperature until the reaction was complete by TLC analysis. To quench excess phosphine, ethanol was added and the reaction mixture was stirred for a further 30 min and dried on a rotary evaporator. The product was purified on silica gel to afford 0.8 g of phosphoramidite 8. 31 P NMR (CDCl 3 , 121.5MHz) δ147.0(s).

Embodiment 3

[0093] Example 3. Conjugated oligonucleotide synthesis (Table 1 and image 3 )

[0094] 2'-ACE protected RNA oligonucleotides (ODN-1.1, ODN-2, ODN-3.1, ODN-4, ODN-5, ODN- 7 and ODN-8), using a polystyrene solid support and 2'-bis(acetoxyethoxy)-methyl ether (2'-ACE) phosphoramidite. For ODN-2 and ODN-4, Aminomethylated polystyrene support 6 was used (see Example 1). For ODN-5, 5'-hexyne phosphoramidite 8 was used. After completion of the synthesis cycle, the oligonucleotides on the support were placed on Na at room temperature 2 S 2 solution, followed by washing with water. Oligonucleotides were cleaved from the support with 40% aqueous N-methylamine (NMA), heated at 55°C, and then freeze-dried. Crude RNA was desalted, purified by HPLC, and the identity of the purified samples was confirmed by UPLC and ESI-MS.

[0095] ODN-1.2 and ODN-3.2: After synthesis, NHS azidoacetate (Click ChemicalTools) dissolved in DMF was added to lyophilized 3'-aminoalkyl-modified oligonucleot...

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PUM

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Abstract

The present invention provides synthetic single guide RNAs that comprise two separate functional sequences (commonly known as crRNA and tracrRNA) connected by a linker. These synthetic single guide RNA molecules are useful in gene editing when used with RNA-guided endonucleases such as cas9 in eukaryotic cells. The availability of the synthetic single guide RNAs makes the screening for gene editing in high-through-put format simple and convenient.

Description

field of invention [0001] The present invention relates to the field of gene editing. Background of the invention [0002] For many years, researchers have investigated the use of oligonucleotides to control activity within cells. Among the approaches explored are those relying on antisense technology and RNA interference ("RNAi") technology. Each of these techniques utilizes the ability of oligonucleotides to target one or more regions of one or more other nucleic acids based on the degree of complementarity of related nucleotide sequences. [0003] One area of ​​recent research related to the control of DNA activity is the use of the CRISPR-Cas system. CRISPR-Cas systems utilize proteins that occur naturally in about 40%-60% of bacteria and about 90% of archaea. The binding of naturally occurring CRISPR proteins to certain types of untranslated RNA has been shown to confer resistance to foreign DNA in these prokaryotes. In these prokaryotes, the CRISPR locus consists o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/09C12N15/10
CPCC12N9/22C12N2310/10C12N2310/318C12N2310/3519C12N2310/20C12N15/111C40B40/06
Inventor 何凯章E·M·安德森M·O·德莱尼
Owner 达尔马科恩有限公司
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