Genome editing expression cassette, vector and application thereof

A technology of genome editing and expression cassettes, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of slow research and development, slow progress of genetic engineering, etc., and achieve the effect of broad application prospects and high gene editing activity

Active Publication Date: 2022-05-27
UNIV OF ELECTRONICS SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since larch belongs to gymnosperms, the progress of genetic engineering in gymnosperms is slow, and there is no report of successful genome editing in larch, which leads to the slow development of research on the application of this emerging technology to improve the genetic traits of larch.

Method used

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  • Genome editing expression cassette, vector and application thereof
  • Genome editing expression cassette, vector and application thereof
  • Genome editing expression cassette, vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction and comparison of backbone vector of larch gene-directed modification system

[0047] 1. 35S::STU Cas9_V01, ZmUBQ1::STU Cas9_V01, LarPE004::STU Cas9_V01 backbone vector construction

[0048] The present invention uses the reported pGEL031 (Tang, Single transcript unit CRISPR2.0 systems for robust Cas9 and Cas12a mediated plant genome editing. 2019, Plant Biotechnol J) vector as the starting backbone vector, which is a strong maize promoter Ubiquitin Co-transcription of Cas9 protein and sgRNA unit is started, and this backbone vector is the backbone vector numbered ZmUBQ1::STU Cas9_V01 in this example.

[0049] Using pGEL031 as the backbone, the endogenous promoter LarPE004 gene fragment of larch was amplified from the genomic DNA of larch with LarPE004-F and LarPE004-R (Table 1), and the cauliflower virus promoter 35S was amplified from 35S-F and 35S-R. pTX171 (Tang, A Single Transcript CRISPR-Cas9 System for Efficient GenomeEditing in Plants. 20...

Embodiment 2

[0060] Example 2 Construction of endogenous gene targeting site-directed mutation vectors of different larch gene-directed editing systems

[0061] Target sites were designed for the endogenous genes of Larch, LarbHLH03, LarMYB06, LarCKX01, LarActin, and LarNAC02, and the upstream and downstream primers were synthesized artificially (see Table 2). Take 20uL of the upstream and downstream primers and mix well, denature at 95°C for 10min, and naturally cool and anneal to form the double-stranded DNA of the target site with sticky ends. The annealed fragments and the BsaI-digested backbone vector products of different larch gene-directed editing systems were cloned and ligated by the Golden Gate method. The Golden Gate reaction system is as follows: 10×T4ligase buffer, 2uL; BsaI, 1uL; T4 DNA ligase, 1uL; Larch gene-directed modification system backbone vector digestion product (100ng), 1uL; annealing to different endogenous gene target sites Product (10 mM), 2uL; ddH2O, 13uL. T...

Embodiment 3

[0065] Example 3 Targeted Mutation Activity Detection of Different Larch Gene Targeted Editing Systems

[0066] 1. Sequencing method to detect the editing activity of different larch gene-directed editing systems for endogenous gene loci

[0067] Through the preparation of larch protoplasts and transient transformation system, the targeted site-directed mutation vectors of endogenous genes of different larch gene-directed editing systems were introduced into larch protoplast cells, and protoplast genomic DNA was extracted after culturing for 48 hours. Amplify different target gene loci using the same amount of sequencing primers (Table 3). The amplification system is as follows: 10×Taq Buffer, 2.5uL; Taq DNA Polymerase (2.5U / μL), 0.4uL; dNTPs (10mM), 0.5uL; upstream detection primer F, 0.5uL; downstream detection primer R, 0.5uL; gDNA (100ng / μL), 2uL; ddH2O, 18.6uL. The amplification program was as follows: 95°C, 3 min; (95°C, 30s; 56°C, 20s; 72°C, 40s) × 34 cycles; 72°C, 5mi...

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Abstract

The invention belongs to the field of plant gene editing, and particularly relates to a genome editing expression cassette, a vector containing the expression cassette and application of the vector. The technical problem to be solved by the invention is that no applicable efficient gene editing system exists in plants, especially gymnosperms at present. According to the technical scheme for solving the technical problem, the genome editing expression cassette is provided, the genome editing expression cassette comprises the following elements: a promoter MarPE004, a Cas9 protein coding gene and an sgRNA cloning and transcription unit, and the promoter MarPE004 drives the Cas9 protein coding gene and the sgRNA cloning and transcription unit to express. The expression framework and the corresponding vector provided by the invention can realize efficient gene editing in plants. Experiments show that the gene editing efficiency in the larch protoplast can reach 72.5%, meanwhile, the gene editing gene has high gene editing activity when PAM is NGG, NAG, NGT and other PAM sites, and the gene editing gene has wide application prospects.

Description

technical field [0001] The invention belongs to the field of plant gene editing, and particularly relates to a genome editing expression cassette, a vector comprising the expression cassette, and applications thereof. Background technique [0002] Plant Genome Editing Technology refers to the use of genome editing tools to artificially edit the genomic DNA of plants, and to introduce predetermined mutational forms such as base insertion, deletion, and substitution, so that the plant genomic DNA can be changed in accordance with human wishes. . After more than ten years of exploration and evolution, plant genome editing tools have been developed using the Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated Cas, CRISPR-Cas system. Third-generation tools for plant genome editing. The CRISPR-Cas genome editing tool has the advantages of low experimental cost, easy vector construction, fast detection speed, and strong developability. It is currently the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/55
CPCC12N15/8218C12N9/22Y02A40/22
Inventor 张勇任秋蓉郑雪莲黄兰唐旭
Owner UNIV OF ELECTRONICS SCI & TECH OF CHINA
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