Method for rapidly analyzing construction condition of CRISPR/Cas9 gene editing vector and application

A technology of gene editing and rapid analysis, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of high price and difficulty in large-scale use, and achieve the effect of low cost and good application prospects

Pending Publication Date: 2021-10-22
HUBEI BOYUAN SYNTHETIC BIOLOGY TECH CO LTD
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Solve the existing problems of constructing a large number of CRISPR / Cas9 gene editing vectors, requiring sequencing analysis, using next-generation sequencing technology, high price and difficulty in large-scale use, etc.

Method used

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  • Method for rapidly analyzing construction condition of CRISPR/Cas9 gene editing vector and application
  • Method for rapidly analyzing construction condition of CRISPR/Cas9 gene editing vector and application
  • Method for rapidly analyzing construction condition of CRISPR/Cas9 gene editing vector and application

Examples

Experimental program
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Effect test

Embodiment 1

[0035] In the following example, 100 constructed CRISPR / Cas9 gene editing vectors were selected and mixed in equal proportions to form a gene editing vector library. The specific operation process is as follows:

[0036] 1.1 Primer design

[0037] According to the vector sequence, design the universal primer sequence of the amplified vector as follows:

[0038] ZT-F: 5'-ggagtgagtaccgtgagcTGGACGACAACAAAGACTAG-3';

[0039] ZT-R: 5'-ggaatgcatgctgcatgcTGCCACTTTTTCAAGTTGAT-3';

[0040] The lowercase letters in the above universal primer sequences for vectors are bridge sequences (as shown in SEQ ID NO.1 and SEQ ID NO.2), and the uppercase letters are universal amplification primers for vectors (it should be noted that different gene editing vectors can be Design different vector universal primer sequences), this embodiment according to the sequencing method of PE150, the distance between the designed forward primer and reverse primer is 80bp from the target position.

[0041] 1.2 ...

Embodiment 2

[0060] Mix 100 successfully constructed CRISPR / Cas9 gene editing vector libraries into Agrobacterium, culture for 2 to 3 days, then scrape the Agrobacterium, resuspend all Agrobacterium, and infect the rice callus. After tissue culture, a large number of regenerated positive tissue culture seedlings were obtained, which were numbered according to the arrangement of the PCR 96-well plate: A1-H12.

[0061] 2.1 Primer design

[0062] According to the vector sequence, the primer design method in Example 1 was the same.

[0063] 2.2 Genome Extraction

[0064] Take the regenerated positive tissue-cultured seedlings, take leaves 1-2 cm in size, put them into a 96-deep well plate, add 50 μL of 0.2 M KOH solution and 1-2 grinding beads to each well, and centrifuge 2 mL of Transfer the tubes to the adapters of the fully automatic sample grinder and run for 30-60 seconds until the buffer turns green; then use a row gun to pipette 1 μL of the supernatant into a 96-well PCR plate as a te...

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Abstract

The invention discloses a method for rapidly analyzing the construction condition of a CRISPR/Cas9 gene editing vector, which comprises the following steps: S1, designing a vector universal primer based on a target interval, performing amplifying, constructing a library, and carrying out high-throughput sequencing to obtain sequencing data; S2, performing quality control on the sequencing data obtained in the step S1; S3, splicing reads at two ends of the quality-controlled data obtained in the step S2 to obtain long sequence data; and S4, analyzing the long sequence data obtained in the step S3 according to the characteristics that the carrier sequences before and after the target interval are known and are the same, then extracting the sequence of the target interval, and carrying out statistical analysis, so as to obtain the construction condition of the target. The method is rapid, accurate and low in cost, multi-sample mixed sequencing can be achieved, thousands of gene editing vectors can be identified, the analysis requirement can be met only through the data size of 1G, and therefore the method has good application prospects in the application field of plant, animal or microorganism vector construction.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a method and application for rapidly analyzing the construction of a CRISPR / Cas9 gene editing vector. Background technique [0002] Genome editing technology is an effective way to modify the genome and study gene functions, but before the genome editing technology was discovered, people could only modify the genome through homologous recombination, but this method relies too much on natural Recombination is extremely inefficient and cannot meet the growing scientific research needs of human beings. In this case, gene editing technology came into being. In recent years, the rise of artificial endonuclease (engineered endonuclease, EEN) technology has made gene editing technology simple and feasible, and the commonly used EEN technology is mainly zinc finger nuclease (zincfinger nuclease, ZFN), transcription-activator-like Factor effector nuclease (transcripti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2535/122C12Q2537/165C12Q2563/185C12Q2531/113C12Q2537/143
Inventor 吴磊
Owner HUBEI BOYUAN SYNTHETIC BIOLOGY TECH CO LTD
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