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SgRNA base mismatched target site library and its application

A DNA library and target site technology, applied in the field of gene modification, can solve problems such as changes in the efficiency of recruiting nucleases, changes in the efficiency of recruiting nucleases with fixed sequences, and difficulty in evaluating recognition efficiency.

Active Publication Date: 2016-02-10
PEKING UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Although the primary sequence of the fixed sequence of this method does not change, it is unknown whether the efficiency of nuclease recruitment by the fixed sequence changes when the sequence of the recognition site changes
That is to say, when different sgRNA sequences produce different cleavage efficiencies for the same target site sequence, it is difficult to evaluate whether the change in recognition efficiency is due to the change in the DNA recognition sequence or the fixed sequence caused by the change in the DNA recognition sequence. Changes in the efficiency of nuclease recruitment
Therefore, the accuracy of off-target effect research based on this method needs to be further improved

Method used

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  • SgRNA base mismatched target site library and its application
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  • SgRNA base mismatched target site library and its application

Examples

Experimental program
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Embodiment 1

[0035] Example 1. sgRNA off-target detection system and sgRNA single-base mismatch target site library construction

[0036] The sgRNA off-target detection system used in the present invention is the pSG-targetCloningKit&SingleStrandedAnnealingAssay (BV3100) system provided by Biomics Biotechnology Co., Ltd. (http: / / www.biomics.com). The system consists of three plasmids: the pCas plasmid encodes the cas9 protein, the psgRNA plasmid encodes the sgRNA sequence, and the pTarget plasmid encodes an inactive firefly luciferase reporter gene. In the inactive state, the firefly luciferase reporter gene is divided into two parts separated by a stop codon, a target site for sgRNA, and two 1000-base direct repeat sequences. When the sgRNA / Cas9 complex acts on the target site of the sgRNA to produce a double-strand cut, the DNA repair system in the cell will, on the basis of the direct repeat sequences on both sides of the cut site, through homologous recombination, convert the broken fr...

Embodiment 2

[0098] Example 2, sgRNA activity detection

[0099] Detection of sgRNA activity: The day before the experiment, HEK293 (human embryonic kidney cells) was mixed with 2.5×10 4 The density of cells / well was inoculated into a 48-well cell culture plate, and the medium was DMEM medium containing 2mML-glutamine, 10% fetal bovine serum (Sigma), 100U / ml penicillin and 100ug / ml streptomycin. When the cells grew to 30-50% density, the cell transfection experiment was performed using Lipo2000 (invitrogen) transfection reagent. According to the requirements of the transfection reagent instructions, each culture well was transfected with 200ng of pCas plasmid, 200ng of psgRNA plasmid, 30ng of pTarget plasmid, and 5ng of renillaluciferase control plasmid pRL-TK. Incubate the cells at 37°C CO 2 After culturing in the incubator for 24 hours, the dual luciferase reporter gene detection system (Promega, ReporterAssay System) and a microplate reader (SynergyHT, BioTek, USA) were used to dete...

Embodiment 3

[0100] Example 3. Application of single-base mismatch target site library in the study of sgRNA off-target effects

[0101] Using the method described in Example 2, in cultured HEK293 cells, using the single-base mismatch target site sequence library constructed in Example 1, the off-target effects of two sgRNAs were detected. The experimental results are as follows figure 2 , 3 shown.

[0102] The above experimental results show that using the single base mismatch target site library provided by the present invention can systematically detect the off-target effect when the sgRNA and the target site sequence are mismatched at each position.

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Abstract

The invention provides a single-base mismatched target site DNA library specific to a single sgRNA molecule, and a carrier library generated by connecting the DNA library with a carrier. The invention also provides an application of the DNA library / carrier library in the study on the detection of sgRNA off-target effect.

Description

technical field [0001] The invention belongs to the technical field of gene modification, and in particular relates to a sgRNA base mismatch target site library and an application thereof. Background technique [0002] Gene editing technology is a genome modification technology developed in recent years. It can perform InDel mutation, knock-in, multi-site mutation, small fragment deletion, and fragment replacement at specific sites in the genome. In the field of scientific research, gene editing technology can be used for the rapid construction of model organisms; in the field of agriculture, this technology can be used to transform plant varieties; and in the field of health care, this technology may also achieve the purpose of treating diseases by modifying human genes . Therefore, gene editing technology has extremely broad development prospects and application value. [0003] At present, gene editing technologies mainly include zinc finger nuclease (ZFN), transcription...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/63C12N15/66C12Q1/68C40B40/06C40B40/02C40B50/06
Inventor 杜权郑婷侯英子
Owner PEKING UNIV
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