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Combinational strategy for reducing ransom integration events when transfecting plants

A random integration, plant technology, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, biochemical equipment and methods, etc., can solve problems such as harmful effects on host organisms

Inactive Publication Date: 2020-08-14
LEIDEN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a disadvantage of current transfection methods is the generation of a relatively large number of random DNA integration events in the host genome
Random integration may have unpredictable and / or deleterious effects on the host organism

Method used

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  • Combinational strategy for reducing ransom integration events when transfecting plants
  • Combinational strategy for reducing ransom integration events when transfecting plants
  • Combinational strategy for reducing ransom integration events when transfecting plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0161] Example 1: Preventing DNA Integration in Plants by Combinatorial Inactivation of Plant Factors

[0162] background

[0163] Genetic modification of plants is now routinely performed. Transformation can be performed by a variety of methods and vectors including Agrobacterium tumefaciens. Transgenes have been observed to integrate in variable copy numbers at fairly random locations in the plant genome by non-homologous recombination (NHR). This can lead to position effects (such as transgene silencing) and genetic mutations at the integration site. Targeted DNA integration via homologous recombination (HR) would avoid this adverse effect while opening the possibility of introducing targeted mutations and edits into the genome via HR. This works well in yeast, but is a rare event in the somatic cells of higher plants. Indeed, integration through the NHR is rare in yeast, strongly favoring integration through the HR. An advantageous way of introducing genes into plant ...

Embodiment 2

[0200] Example 2: Homologous recombination

[0201] Example 1 shows that functional mre11 and Ku80 are required for stable random integration of T-DNA. This example demonstrates that stable integration by homologous recombination does not require functional mre11 and Ku80.

[0202] A T-DNA construct was prepared with about 6 kb of homology to the endogenous Arabidopsis locus protophorphyrinogen oxidase (PPO). The region of homology contains two point mutations that, after integration at the PPO locus by homologous recombination, confer resistance to the herbicide butafenicil (for a description of the mutations, see Hanin et al, Plant J 2001). In addition, the T-DNA encodes the Cas9 enzyme (Arabidopsis codon-optimized Cas9-AteCas9 (Fauser et al. Plant J 79:348-3592014)) and a guide RNA that directs the Cas9 enzyme to the PPO locus. The expression of Cas9 is driven by the ubiquitin promoter, and the guide RNA is under the control of the U6 (AtU6) promoter.

[0203] Wild-type...

Embodiment 3

[0205] Example 3: Homologous recombination

[0206] A combination of functional Pol θ and / or Ku80 is required for stable random integration of the T-DNA. This example demonstrates that stable integration by homologous recombination does not require functional Pol θ and / or Ku80.

[0207] A T-DNA construct was prepared with approximately 6 kb of homology to the endogenous Arabidopsis locus protoporphyrinogen oxidase (PPO). The region of homology contains two point mutations that, after integration at the PPO locus by homologous recombination, confer resistance to the herbicide flumethazine (see Hanin et al, Plant J 2001 for a description of the mutations) . In addition, the T-DNA encodes the Cas9 enzyme (Arabidopsis codon-optimized Cas9-AteCas9 (Fauser et al. Plant J 79:348-3592014)) and a guide RNA that directs the Cas9 enzyme to the PPO locus. The expression of Cas9 is driven by the ubiquitin promoter, and the guide RNA is under the control of the U6 (AtU6) promoter.

[02...

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Abstract

The present disclosure provides methods for transfecting plants and for expressing RNA or polypeptide molecules in plants. In particular, plants having reduced expression and / or activity of a component of the classical NHEJ pathway and a component of the POLQ pathway are transfected in order to reduce random integration events. The disclosure further provides transfected plants and plant progeny produced by the methods disclosed herein.

Description

technical field [0001] The present disclosure provides methods for transfecting plants and expressing RNA or polypeptide molecules in plants. In particular, plants are transfected with reduced expression and / or activity of canonical NHEJ pathway components and POLQ pathway components to reduce random integration events. The present disclosure also provides transfected plants and plant progeny produced by the methods disclosed herein. Background technique [0002] Genetic modification of plants by transfection can alter traits such as increased yield, disease and pest resistance, increased vegetative biomass, herbicide tolerance, nutritional quality, drought and stress tolerance, and horticultural qualities such as Pigmentation and growth and other agronomic traits for crop improvement. In addition, genetic modification of plants has been used as a system for expression of recombinant proteins. [0003] Transfection of plants with nucleic acids is now a common practice and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
CPCC12N15/8213C12N15/8218
Inventor 保罗·扬·雅各布·霍伊卡斯贝纳德特·西尔维娅·德帕特尔马塞尔·蒂斯特曼
Owner LEIDEN UNIVERSITY
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