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Method for rapidly constructing CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) gene editing vector library

A gene editing and vector technology, applied in the field of molecular biology, can solve the problems of low connection efficiency and short double-stranded DNA length, and achieve the effect of ensuring uniformity

Inactive Publication Date: 2019-06-07
CHINA TOBACCO YUNNAN IND
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Problems solved by technology

However, due to the short length of double-stranded DNA generated by the double-stranded annealing method, the ligation efficiency is low during library construction

Method used

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  • Method for rapidly constructing CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) gene editing vector library
  • Method for rapidly constructing CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) gene editing vector library
  • Method for rapidly constructing CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) gene editing vector library

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Embodiment 1

[0025] Example 1 CRISPR / Cas9 gene editing vector library construction.

[0026] Such as figure 1 As shown, the CRISPR / Cas9 gene editing vector library construction method of the present embodiment is characterized in that: comprising the following steps:

[0027] In step (1), 402 DNA oligonucleotides (SEQ ID No.1-402) were artificially synthesized for 201 editing sites, and the length of each sequence was 25nt.

[0028] Step (2), annealing the double strands of 402 artificially synthesized DNA oligonucleotides, as follows:

[0029] Dilute 402 DNA oligonucleotides to 100 ng / μl with sterile ultrapure water, pipette 5 μl of upstream and downstream primers into 200 μl PCR tubes, transfer to PCR instrument for annealing, and then add sterile ultrapure water to dilute to 100 μl.

[0030] The annealing program is: 95°C for 5min, -0.1°C / 8s, annealing to 25°C. Then transfer it to a centrifuge tube at a ratio of 1:1 to form the sgRNA library.

[0031] Step (3), BsaI digestion of the...

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Abstract

The invention provides a method for rapidly constructing a CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR associated protein 9) gene editing vector library. The method comprises the following steps: designing specific sgRNA (small guide Ribonucleic Acid); artificially synthesizing oligonucleotide DNA (Deoxyribonucleic Acid); adding cohesive terminus DNA basic groupsat 5' ends of upstream and downstream primers respectively; carrying out double-strand annealing on DNA oligonucleotide to form double-strand sgRNA; connecting the double-strand sgRNA to an enzyme digestion linear CRISPR / Cas9 plasmid by utilizing T4 / T7 DNA ligase; and after transforming escherichia coli through a connection product to extract plasmids, obtaining the editing vector library. The invention constructs a method for constructing gene editing vectors in a large scale by utilizing a double-strand annealing principle; the method has very good accuracy, coverage degree and uniformity; the construction of hundreds and thousands of the gene editing vectors can be realized through one-time operation and the construction efficiency of the vectors is greatly improved; and the method hassignificant meaning on researches of various species genes and germplasm resource development.

Description

technical field [0001] The invention relates to a method for rapidly constructing a gene editing vector library, in particular to a method for rapidly constructing a CRISPR / Cas9 gene editing vector library, and belongs to the field of molecular biology. Background technique [0002] Since 2012, a CRISPR / Cas9-based gene editing technology (hereinafter referred to as gene editing) has been widely used. It has simple principles, efficient editing, and low cost. It has become a mainstream biotechnology in basic research and application of animals and plants. [0003] The key to gene editing technology is to insert a 20bp sgRNA guide sequence. The current mainstream insertion methods include overlapping PCR method and double-strand annealing method. The overlapping PCR method has been reported in many literatures. However, the poor uniformity of the PCR amplification process is prone to biased amplification, which makes the number of some sgRNAs dominant, making it very difficul...

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Application Information

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IPC IPC(8): C40B50/06C40B40/06C40B40/08C12N15/70
Inventor 张建铎李雪梅陈章玉杨光宇向海英曾婉俐张涛高茜宋春满许力蒋佳芮邓乐乐杨文武贾凌夏庆友
Owner CHINA TOBACCO YUNNAN IND
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