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Optimized plant CRISPR/CPF1 systems

A plant and delivery system technology, applied in the field of optimized systems and plant delivery systems, can solve the problem of low predictability

Pending Publication Date: 2020-10-27
KWS SAAT SE & CO KGAA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite recent advances in the field, the predictability of the success of GE experiments planned in silico remains low, especially for plant genomes because much of the CRISPR work is performed in animal cells, and the complexity of plant genomes and specific traits as well as plant metabolism still require fundamental research work to establish efficient CRISPR systems for a wide variety of different plants, including economically relevant crops

Method used

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  • Optimized plant CRISPR/CPF1 systems
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  • Optimized plant CRISPR/CPF1 systems

Examples

Experimental program
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Embodiment 1

[0259] Example 1: Target Sequence

[0260] For the purpose of this analysis, various gene editing plasmids (GEPs) were designed and used. As shown below, Table 1 provides the internal designation (crGEP) of the different gene editing plasmids (left column), information about the target gene (middle column), and the respective target sites of selected crRNAs or sgRNAs (right column). The target site represents the actual binding site in the genomic target region or gene of interest.

[0261] Table 1:

[0262]

[0263]

Embodiment 2

[0264] Embodiment 2: plant protoplast transfection

[0265] When transfecting plant protoplasts for the experimental purposes disclosed herein, protocols known in the art were used according to the following steps:

[0266]The buffers and solutions used are enzyme liquid, enzyme wash, enzyme wash buffer (EWB), MMG (ethylene glycol-magnesium mannitol) buffer, etc., which contain 0.1 to 0.5M mannitol, 15mM to 20mM magnesium chloride and 4 mM MES (pH 10 to 40% PEG (polyethylene glycol) calcium, stop buffer and W5 buffer (for example, containing 154 mM sodium chloride, 125 mM calcium nitride, 5 mM potassium chloride, 2 mM M ES (pH 5.7)) glucose ).

[0267] First, 20 μg (or unless otherwise specified) of plasmid DNA was added to a 2 ml tube at 4°C. Second, harvest fully expanded first and second leaves from 10-14-day-old etiolated seedlings in a true-leaf greenhouse and place them in a bag with wet paper towels. The leaves were cut into thin strips weighing 4.5 g. Place in a de...

Embodiment 3

[0272] Example 3: Next Generation Sequencing (NGS) Protocol

[0273] When using next-generation tests for experiments, the following protocols are followed: Library preparation: Libraries are prepared by two PCR steps to amplify regions of interest and add sequencing adapters. Barcodes were designed with primers and added in the first PCR step for sample identification. Adapters are added during the second PCR for sequencing. Next-generation sequencing (NGS): The amplicons were sequenced using the Illumina Miseq150 PE platform. The sequencing coverage was 100,000-fold for the protoplast population, and 250,000-fold, 300,000-fold, and 50,000-fold for immature embryos and callus, Agrobacterium-transformed leaf samples, and bombarded wheat leaf samples, respectively. Analyze data using FastQC+Jemultiplexer+Trimmomatic for read QC and demultiplexing, use CRISPResso for identification of target gains and losses, and edit event calls via internal impact client scripts. The entire...

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Abstract

The present invention relates to optimized systems for the genome editing of eukaryotic cells, preferably plant cells. Also provided is a plant delivery system comprising at least one Cpf1 enzyme, ora plant optimized construct encoding the same, to be synergistically combined with a Cpf1 guide RNA system. Further provided is a Cpf1 guide RNA system being flanked by a Hammerhead ribozyme sequenceat the 5' end and by a plant-derived Hepatitis Delta Virus (HDV)-like ribozyme sequence at the 3' end, or being embedded within a coding or non-coding region, of a sequence encoding a frame sequence.Novel plant-derived HDV-like ribozyme sequences are also provided. Further provided are methods for improved genome editing, and the use of the various systems provided herein to obtain transformed plants, plant cells, tissues, organs, or a progeny thereof, or a plant material, modified in a targeted way even at difficult to access target sites.

Description

technical field [0001] The present invention relates to an optimized system for genome editing in eukaryotic cells, preferably plant cells. The present invention also provides a plant delivery system comprising at least one Cpf1 enzyme or a plant-optimized construct encoding it for synergistic combination with a Cpf1 guide RNA system. The present invention also provides a Cpf1 guide RNA system flanked by a hammerhead ribozyme sequence at the 5' end and a plant-derived hepatitis D virus (HDV)-like ribozyme sequence at the 3' end, or embedded in the coding frame In the coding or non-coding region of the sequence (framesequence). The present invention also provides novel plant-derived HDV-like ribozyme sequences. The present invention also provides improved genome editing methods, and the various systems provided by the present invention are useful in obtaining transformed plants, plant cells, tissues, organs or other genes modified in a targeted manner even at difficult-to-rea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/113C12N15/82A01H6/46A01H6/02
CPCC07K14/415C12N15/8213C12N15/8216C12N15/8218A01H6/4684A01H6/024C12N2310/20C12N9/22C12N15/82
Inventor A·赫梅尔Z·瓦格齐帕瓦拉
Owner KWS SAAT SE & CO KGAA
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