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Application of CRISPR/xcas9 gene editing system in cotton

A gene editing, phse401-xcas9 technology, applied in the field of efficient genome editing of upland cotton, can solve the problems of limited PAM sequence and limited range of Cas9 target selection

Active Publication Date: 2022-07-22
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] SpCas9 nuclease can carry out effective gene editing in a variety of cells and organisms, and can recognize the classic 5'-NGG-3'PAM sequence, but this PAM sequence is limited in both mammals and plants, so This greatly limits the range of target selection for Cas9

Method used

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  • Application of CRISPR/xcas9 gene editing system in cotton
  • Application of CRISPR/xcas9 gene editing system in cotton
  • Application of CRISPR/xcas9 gene editing system in cotton

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Obtaining pHSE401-xCas9 vector

[0031] The target sequence xCas9 was synthesized into the vector pUC57 by Shanghai Shenggong, and named as: pUC57-xCas9, the xCas9 sequence is the 3224 bp-7324 bp in SEQ ID NO. The carrier pHSE401-Cas9 ( figure 1 ), the pHSE401-xCas9 vector was obtained.

[0032] The specific operations are as follows: (1) Analyze the pHSE401-Cas9 vector, select the appropriate double restriction sites Xba I and Sac I, and design the homology arms and restriction sites sequences containing about 20 bp bases on the vector at both ends of the gene primer F / R. xCas-F / R, according to the amplification system in Table 1, and the reaction program in Table 2 to obtain the xCas9 fragment, and the amplification primer sequences are:

[0033] xCas-F: 5'-CGATACTCGAGTAATCTAGATGGATTACAAGGACCACGA-3' (SEQ ID NO. 2)

[0034] xCas-R: 5'-CGAACGAAAGCTCTGAGCTCACTTCTTTTTCTTAGCCTGTCCGG-3' (SEQ ID NO. 3)

[0035] Table 1 TKS amplification system

[0036]

[...

Embodiment 2

[0051] Example 2: Construction of gene editing vector based on U6-sgRNA as backbone

[0052] The exogenous GFP protein was used as the sgRNA target gene, and the sgRNA sequence of the GFP gene was predicted according to the CRISPRscan online analysis tool (http: / / www.crisprscan.org / ?page=sequence), and the sgRNA sequence of the GFP gene was predicted according to the online software (http: / / www. rgenome.net / cas-offinder / ) to analyze the off-target efficiency of the target sequence in cotton, and finally select the sgRNA sequence that is highly edited, and its sequence is:

[0053] sgEGFP-1: 5'-ATACCCAGATCATATGAAG-3' (SEQ ID NO. 4)

[0054] sgEGFP-2: 5'-CAGCTGCTGGGATTACACA-3' (SEQ ID NO. 5)

[0055] Using the pCBC-DT1T2 vector as a template, four-primer PCR amplification was performed to obtain the target sequence (the sequence is shown in SEQ ID NO. 6), and finally the sgRNA target gene expression cassette (such as figure 2 shown). Among them, the amplification primers are...

Embodiment 3

[0074] Example 3: Detection of editing efficiency in protoplasts

[0075] 1. Culture of GFP transgenic seedlings:

[0076] The cotton seeds were placed in the medium at a temperature of 25±2°C, and cultivated in the dark for about 10 days.

[0077] 2. Protoplast isolation:

[0078] 1) Take the young leaves of cotton, cut the middle part of them into 0.5-1mm filaments with a blade, put them into 50ml of enzyme solution, vacuum pump for 30min, pressure about 15kpa, take out and let stand for digestion for 5h.

[0079] Note: The enzymolysis temperature should be kept at 20-25℃, and it should be protected from light. After the enzymolysis, gently shake the enzymolysis solution to release the protoplasts.

[0080] 2) Add 50 ml of W5 solution to dilute the enzymatic hydrolysis product, and filter the enzymatic hydrolysis solution with a 75 μm nylon filter into a round-bottom centrifuge tube (50 ml).

[0081] Note: The nylon membrane should be soaked in 75% (v / v) alcohol, rinsed w...

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Abstract

The invention discloses the application of the CRISPR / xCas9 gene editing system in cotton. The technical scheme is that the invention synthesizes the target sequence xCas9 in Shanghai Shenggong, and clones it into the target vector pHSE401-Cas9 through homologous arm recombination to obtain an efficient editing vector pHSE401-xCas9, its sequence is shown in SEQ ID NO.1. At the same time, the present invention verifies the editing efficiency of the vector pHSE401-xCas9 in cotton protoplasts and callus. The results show that the present invention verifies that the editing efficiency of pHSE401-xCas9 in cotton protoplasts is higher than that of pHSE401-Cas9; The editing efficiency of xCas9 is higher than that of pHSE401‑Cas9. The present invention successfully establishes the CRISPR / xCas9 gene editing system for the first time in cotton, the system shows high editing efficiency, and will become a new important technical means for cotton functional genome research.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, in particular to an efficient editing method for upland cotton genome. Background technique [0002] CRISPR / Cas mainly exists in archaea and bacteria, and it is an acquired immune defense mechanism that bacteria and archaea continuously evolve in the process of resisting the invasion of foreign viruses and phages. At present, the CRISPR / Cas9 system is the most widely used and mature gene editing system, and has been widely used in cell gene editing of animals and plants. mutants to study gene function. [0003] SpCas9 nuclease can perform efficient gene editing in a variety of cells and organisms, and can recognize the classic 5'-NGG-3' PAM sequence, but this PAM sequence is limited in both mammals and plants, so This greatly limits the target selection range of Cas9. The variant of Cas9, xCas9 3.6, can recognize three patterns of PAM sequences, 5'-NG-3', 5'-GAA-3' and 5'-GAT...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/66C12N15/55A01H5/00A01H6/60
CPCC12N15/8218C12N15/66C12N9/22
Inventor 詹晶晶葛晓阳杨召恩王鹏陈艳丽
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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