Methods and compositions for homology directed repair of double strand breaks in plant cell genomes

A double-strand break and homologous recombination technology, applied in the field of molecular biology, can solve problems such as low specificity, high preparation cost, and time-consuming

Pending Publication Date: 2020-12-15
PIONEER HI BRED INT INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Genome editing technologies such as designer zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or homing meganucleases can be used to generate targeted genome interference, but these systems tend to have low specificity are specific and use designer nucleases that require redesign for each target site, making their preparation costly and time-consuming

Method used

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  • Methods and compositions for homology directed repair of double strand breaks in plant cell genomes
  • Methods and compositions for homology directed repair of double strand breaks in plant cell genomes
  • Methods and compositions for homology directed repair of double strand breaks in plant cell genomes

Examples

Experimental program
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example 1

[0496] Example 1: Vector construction

[0497] Maize vector for SDN3

[0498] The T-DNA vector used in these experiments was Figure 5-8 Vectors 1-4 shown in and given as SEQ ID NOs: 46-49, respectively.

[0499]Each vector contains morphogenic factors (WUS and ODP2 under the Axig and PLTP promoters, respectively), cas9 driven by the ubiquitin promoter, and a target site operably linked to the maize U6 polymerase III promoter SEQ ID NO: 17 gRNA of dot (TS) sequence (SEQID NO:30), and two selectable marker genes - herbicide resistance ALS under native maize ALS and ubiquitin promoters (SEQID NO:20 and SEQID NO:8), respectively (HRA SEQ ID NO: 21) and neomycin phosphotransferase II (NptII, DNA SEQ ID NO: 24). The nptII gene driven by the ubiquitin promoter is flanked by a DNA fragment homologous to the sequence at the TS region (HR arms SEQ ID NO: 32 and SEQ ID NO: 33 for genotype A; HR arm SEQ ID NO for genotype B : 34 and SEQ ID NO: 35; for the HR arm of genotype C, SEQ ...

example 2

[0511] Example 2: Cell Transformation

[0512] Methods of Agrobacterium-mediated transformation of cells are known in the art (see, eg, US Patent Nos. 5,563,055 and 5,981,840). In one example, the method described in US 20170121722 A1 (published May 4, 2017) was used.

[0513] Prepare the Agrobacterium master plate.

[0514] Master plates were made by streaking Agrobacterium tumefaciens with binary donor vectors into solid 12V medium from -80°C frozen aliquots and incubating at 28°C for 2–3 days in the dark.

[0515] Grow Agrobacterium on solid media.

[0516] Pick single or multiple Agrobacterium colonies from the master plate and streak them onto a second plate containing 810I medium and incubate for 1-2 days at 28 °C in the dark. Add Agrobacterium infection medium (700 medium; 5 ml) and 100 mM 3′-5′-dimethoxy-4-hydroxyacetophenone (acetosyringone; 5 μL) to a 14 mL conical tube in a fume hood . About 3 full rings of Agrobacterium from the second plate were suspended...

example 3

[0529] Example 3: Plant regeneration

[0530] maize regeneration

[0531] Sixteen days later, embryos with healthy somatic embryos produced in Example 2 were transferred to regeneration medium.

[0532] In one example, the embryos were treated with Agrobacterium, and the selected embryos were transferred to 605T medium (not selected in the first week) one day later, with 0.1 mg / l ethametsulfuron-methyl containing AA in the 605T medium ( Early selection with AA) or in 605T medium with 0.1 mg / l ethametsulfuron (early selection without AA). For the next transfer, the selected embryos were moved to their respective maturation medium. For eventual transfer to rooting medium, single event selected seedlings were removed. For this experiment, the total time elapsed from Agrobacterium infection to the greenhouse was 48 days.

[0533] In another example, embryos were treated with Agrobacterium in liquid for 5 minutes and then co-cultured on 710I medium for one day. At this point...

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Abstract

Methods and compositions are provided for the improvement of homology-directed repair of a double strand break in a plant cell, via the use of a polynucleotide comprising sequences homologous to the target site. In some aspects, the double strand break is created by an RNA-guided Cas endonuclease. The homology-directed repair of the double-strand break may include incorporation of a heterologous polynucleotide, for example a gene encoding a trait of agronomic importance. The homology-directed repair of the double-strand break may occur as a result of template-directed repair using a polynucleotide repair template.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 667968, filed May 7, 2018, and U.S. Provisional Patent Application Serial No. 62 / 751845, filed October 29, 2018, all of which are hereby incorporated by reference The entire content is incorporated here. [0003] References to Sequence Listings Submitted Electronically [0004] The official copy of this Sequence Listing is submitted electronically via EFS-Web as a Sequence Listing in ASCII format, filename 7781WOPCT_SequenceListing_ST25.txt, created on May 01, 2019 and has a size of 1,009,240 bytes, and is submitted simultaneously with this specification. The Sequence Listing included in this file in ASCII format is part of this specification and is hereby incorporated by reference in its entirety. technical field [0005] The present disclosure relates to the field of molecular biology, in particular to compositions and methods fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C12N5/10C12N9/22C12N15/09C12N15/11C12N15/82
CPCC12N15/102C12N15/902C12N2310/20C12N15/8213C12N15/111C12N9/22C12N2800/80
Inventor A·阿南德W·J·戈登-卡姆S·库马刘占斌S·斯维塔舍夫
Owner PIONEER HI BRED INT INC
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