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Double gRNA, double gRNA library, and double gRNA vector library, preparation method and applications thereof

A library and carrier technology, applied in the field of genetic engineering, can solve problems such as low knockout efficiency, difficulty in replying experiments, and destruction of open reading frames

Inactive Publication Date: 2017-07-21
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many lncRNAs are located in the nucleus, which greatly increases the difficulty of knocking out lncRNAs by RNA interference
At the same time, RNAi has the following disadvantages: (1) It is difficult to carry out "reversion experiment" because the exogenous counterpart is also constrained by the RNAi reagent; (2) The knockout efficiency is not high, and high levels of false negative expression are prone to occur , especially in shRNA vectors
[0007] Given the high efficiency of the CRISPR / Cas9 system, the knockout of protein-coding genes is relatively easy, because a small base deletion or insertion can destroy the open reading frame, resulting in non-expression of functional gene products
However, the situation of non-coding genes, especially lncRNAs, is very different. A small base deletion or insertion does not necessarily lead to the loss of lncRNA function. How to further improve the CRISPR / Cas9 gene editing technology to achieve The efficient knockout of all genes or genetic elements is a problem that needs to be considered and solved in current research

Method used

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  • Double gRNA, double gRNA library, and double gRNA vector library, preparation method and applications thereof
  • Double gRNA, double gRNA library, and double gRNA vector library, preparation method and applications thereof
  • Double gRNA, double gRNA library, and double gRNA vector library, preparation method and applications thereof

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Experimental program
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Effect test

Embodiment 1

[0061] The construction of embodiment 1 double gRNA carrier library

[0062] There are more than 50,000 lncRNAs listed in the non-coding database (www.http: / / noncode.org / ). In principle, there is no upper limit for the construction of the double gRNA carrier library of the present invention, but considering that the current U.S. customarray company uses "custom chips" The upper limit of the number of single-stranded oligonucleotides synthesized by the method, we screened about 45,000 lncRNAs suitable for sgRNA labeling, and designed two double gRNAs for each lncRNA to constitute a total of 9,000 double gRNA KO libraries. In addition, 100 non-human gene negative control double gRNAs were also set up.

[0063] The construction method of the double gRNA KO library in this embodiment (the construction process is as follows figure 1 shown) include:

[0064] (1) Preparation of mixed single-stranded oligonucleotide library

[0065] Entrust the American customarray company (http: / / ...

Embodiment 3

[0092] The gene knockout efficiency comparison of embodiment 3 single gRNA and double gRNA

[0093] This embodiment takes the UCA1 gene with 3 exons as an example to compare the gene knockout efficiency of sgRNA and double gRNA (such as image 3 ).

[0094] (1) Gene knockout efficiency of double gRNA

[0095] With the UCA1 gene as the target gene, select the target site on the target gene (such as image 3 As shown in part D), design UCA1-dual gRNA, the base sequences of the positive-sense strands of the two gRNAs in UCA1-dual gRNA are:

[0096] UCA1-gRNA1 (SEQ ID No.8): 5'-GTGCATGGTGGAGAGATGAT-3';

[0097] UCA1-gRNA2 (SEQ ID No.9): 5'-TTCTGGAATGGTGAACCCAA-3';

[0098] Entrust the American genscript company to construct the expression vector containing hCas9 and UCA1-dual gRNA (each DNA content is 0.5 μg), and use the expression vector to transfect and knock out 293T cells. The knockout steps are as follows:

[0099] 1) Subculture 293T cells, and maintain the cell density...

Embodiment 4

[0135] Example 4 Application of double gRNA KO library

[0136] Although the role of long noncoding RNAs (lncRNAs) has been increasingly recognized in various physiological and pathological conditions, the potential roles of large numbers of lncRNAs in breast cancer are less understood. We investigated the expression profile of lncRNAs by RT-PCR and identified a set of lncRNAs that were differentially expressed in patient breast cancer tissues relative to normal breast tissues, respectively. Among them, AK023948 is up-regulated in breast cancer, and it is also up-regulated in breast cancer cell lines (MCF-7 and MDA-MB-231) compared to human breast epithelial cells (HMLE). In situ hybridization was further validated.

[0137] In this embodiment, AK023948 is used as the target gene, and the target site is selected on the target gene (such as Figure 5 As shown in part A), design AK023948-dual gRNA, the base sequences of the sense strands of the two gRNAs in AK023948-dual gRNA ...

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Abstract

The invention discloses a double gRNA, a double gRNA library, and a double gRNA vector library, a preparation method and applications thereof. The double gRNA is formed by sequentially linking a first promoter, gRNA-a, a crRNA scaffold sequence, a second promoter, and gRNA-b. The double gRNA library comprises a plurality of double gRNAs for a target gene. The double gRNA vector library is formed by linking the double gRNA library and a vector. According to the present invention, in the double gRNA, the independent expressions of the gRNA-a and the gRNA-b are controlled respectively by different promoters, and the gRNA-a and the gRNA-b are respectively complemented and paired with two target sites on the target gene, such that the double gRNA can recruit the Cas9 nuclease to cleave the target gent at the two sgRNA action sites so as to effectively improve the knockout efficiency of the target gene.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a double gRNA, a double gRNA library, a double gRNA carrier library and a preparation method and application thereof. Background technique [0002] It is well known that the transcription of protein-coding genes only accounts for about 2% of the whole genome transcription, and the rest of the transcription is the transcription of non-coding RNA such as microRNA and lncRNA. Among them, lncRNA is defined as a group of non-coding RNAs with a molecular weight greater than 200 nucleotides in length. More and more studies have shown that lncRNA may serve as the main gene regulator in various mechanisms, and the disorder of lncRNA expression is usually associated with a variety of Human diseases, such as cancer, are closely related. [0003] So far, more than 50,000 lncRNAs have been discovered, far more than the number of protein-coding genes, providing further evidence ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C40B40/06C40B50/06C40B40/02
CPCC12N15/113C40B40/02C40B40/06C40B50/06C12N2310/10
Inventor 丁先锋莫寅元
Owner ZHEJIANG SCI-TECH UNIV
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