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Eukaryotic gene editing method based on gene cas7-3 in I type CRISPR-Cas system

A gene editing and gene technology, applied in the field of genetic engineering and eukaryotic gene editing, to achieve the effect of effective gene editing

Pending Publication Date: 2018-01-09
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although we have reported the use of type I CRISPR-Cas systems containing multiple Cas effectors for gene editing in prokaryotes (Tong Wangyu, Xu Xin, Zhang Yan, Sun Yan, Cao Suli, a Streptomyces virginia IBL14type I-B-sv14 type CAS gene editing system, CN201611113137.3; Tong Wangyu, Qiu Caihua, Yang Xingwang, Wang Jingjing, a gene editing method based on the cas7-5-3 gene of Streptomyces virginia IBL14, CN201611089333.1), However, the application of eukaryotic gene editing has not been reported

Method used

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  • Eukaryotic gene editing method based on gene cas7-3 in I type CRISPR-Cas system
  • Eukaryotic gene editing method based on gene cas7-3 in I type CRISPR-Cas system
  • Eukaryotic gene editing method based on gene cas7-3 in I type CRISPR-Cas system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 pRS415-cas7-3 and pYES2 / NTA-t / g-ΔcrtI were respectively transformed to knock out the SC BY4741 gene crtI

[0049] (1) Construction of protein expression plasmid pRS415-cas7-3

[0050] (A) Construction of protein expression plasmid pRS415-cas3

[0051] According to the sequence information of plasmid pRS415, specific primers cas3-F / cas3-R and pRS415-cas3-F / pRS415-cas3-R were designed, and the two ends of the primers carried the complementary sequences of cas3 gene and plasmid pRS415-cas9 respectively. Cas3 gene PCR amplification was performed using TransStart FastPfu DNA Polymerase produced by Quanshijin Biotechnology Co., Ltd., and the reaction conditions were: 95±1°C for 5±1min, 95±1°C for 30s, 62±3°C for 30s, and 72°C for 1±1min ( 50μl reaction system), 30 cycles, 72°C for 10min. The PCR product was detected by 1% agarose electrophoresis, recovered by the kit, and a purified cas3 gene fragment was obtained. The same PCR amplification was performed again t...

Embodiment 2

[0073] Example 2 pRS415-cas7-3 and pYES2 / NTA-t-ΔcrtI were respectively transformed to knock out the SC BY4741 gene crtI

[0074] (1) Construction of protein expression plasmid pRS415-cas7-3

[0075] With embodiment 1 step (1)

[0076] (2) Construction of gene editing template vector pYES2 / NTA-t-ΔcrtI

[0077] (A) Preparation of upstream and downstream homology arms

[0078] With embodiment 1 step (2A)

[0079] (B) Construction of gene editing template vector pYES2 / NTA-t-ΔcrtI

[0080] With embodiment 1 step (2B)

[0081] (3) Acquisition and inspection of recombinants

[0082] (A) Competent preparation of SCBY4741 and transformation of pRS415-cas7-3

[0083] With embodiment 1 step (3-A)

[0084] (B) Competent preparation of SC BY4741-pRS415-cas7-3 and transformation of pYES2 / NTA-t-ΔcrtI

[0085] Except importing plasmid pYES2 / NTA-t-ΔcrtI, all the other are the same as embodiment 1 step (3B) (see figure 2 ).

Embodiment 3

[0086] Example 3 Transformation of single plasmid pRS415-cas7-3-t / g-ΔcrtI to knock out SC BY4741 gene crtI

[0087] (1) Construction of protein expression plasmid pRS415-cas7-3

[0088] With embodiment 1 step (1)

[0089] (2) Construction of protein expression and gene editing single plasmid pRS415-cas7-3-t / g-ΔcrtI

[0090] (A) Preparation of upstream and downstream homology arms

[0091] Except that crtI gene upstream homology arm primer crtI-UF2, downstream homology arm primer crtI-DR2 contain XhoI restriction enzyme site simultaneously, other is the same as embodiment 1 step (2A), used upstream homology arm primer crtI-UF2 The sequence of the primer crtI-DR2 is ccgCTCGAGaccaactgaacgagcaataacgg.

[0092] (B) Construction of protein expression and gene editing single plasmid pRS415-cas7-3-t / g-ΔcrtI

[0093] Except that the g-crtI-DNA and t-ΔcrtI-DNA fragments were respectively connected to the pRS415-cas7-3 plasmid, other steps were the same as step (2B) of Example 1.

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Abstract

The invention discloses gene knockout and gene insertion on eukaryotic genomes carried out by two Cas proteins (Cas7 and Cas3) in a 1 class I type CRISPR-Cas system for the first time. The developmentof the method breaks the limitation of dependence on single-gene cas9 and cpf1, and provides a new perspective for performing gene editing on the eukaryotes by multi-gene. By applying the system, thegene editing can be conveniently, rapidly and effectively carried out on eukaryote brewer's yeast genomes. The optimized tool is expected to be widely used in the gene editing of other eukaryotes.

Description

[0001] The invention relates to the genetic engineering technology in the field of biotechnology, specifically a eukaryotic gene editing method based on the gene cas7-3 in the type I CRISPR-Cas system. Background technique [0002] Genetic engineering, or DNA recombination technology, is a revolutionary technology based on restriction endonuclease and ligase. It usually refers to the technology of integrating genes from different sources into vectors (such as plasmids or viruses) in vitro according to a pre-designed blueprint, and then introducing them into living cells to change the original genetic characteristics of organisms, obtain new varieties or increase product yield. Its characteristic is that the modification and function realization of the DNA are mainly realized through the engineering plasmid and the engineering virus capable of self-replicating. Genome editing technology (referred to as gene editing technology) is the same as genetic engineering that is often sa...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/82C12N15/85C12N15/90
Inventor 童望宇夏婷婷唐严严
Owner ANHUI UNIVERSITY
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