CRISPR Plant Cell Editing via PEG-Mediated Delivery
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Solution Overview
Problem
Current methods for targeted genome editing in plant cells using CRISPR-based systems are not always successful or efficient, and existing methodologies optimized for animal cells are not effective when applied to plant cells, necessitating the development of new compositions and methods for reliable, efficient, and reproducible targeted DNA alteration in plant cells.
Innovation Solution
A method involving an aqueous medium comprising a CRISPR-associated protein (Cas protein) and a CRISPR-CAS system guide RNA, with polyethylene glycol (PEG) and minimal or no glycerol, specifically optimized for plant cells, which includes parameters such as temperature, duration of contact, and concentrations of Cas protein and sgRNA to enhance targeted DNA alteration efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR-based genome editing methods are applied to plant cells using protocols optimized for animal cells, then the methodology can be transferred to a new system, but the efficiency and success rate of targeted DNA alteration deteriorates
Solution Approach 1:
The patent optimizes multiple parameters including PEG concentration (10-40% w/v), glycerol concentration (0-20% v/v), incubation temperature (0-37°C), and incubation time (1-24 hours) to achieve effective CRISPR-mediated genome editing in plant cells, transforming the protocol from animal-cell-optimized to plant-cell-optimized conditions
2Reliability
If traditional plasmid-based methods are used for genome editing in plant cells, then the process is well-established, but the time required for transformation and selection increases
Solution Approach 1:
The patent performs CRISPR-mediated genome editing directly in plant protoplasts without requiring plasmid transformation, selection, and regeneration steps. The Cas9 protein and guide RNA are delivered directly to protoplasts, enabling immediate editing action and skipping multiple time-consuming intermediate steps
3Ease of operation
If CRISPR components are introduced into plant cells using standard transfection protocols, then the delivery method is simple, but the protoplast survival rate deteriorates
Solution Approach 1:
The patent identifies and optimizes critical parameters including PEG concentration (10-40% w/v), glycerol concentration (0-20% v/v), incubation temperature (0-37°C), and incubation time (1-24 hours) to achieve effective CRISPR-mediated genome editing while maintaining high protoplast survival rates
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach achieves effective targeted alteration of plant DNA with improved protoplast survival and growth, demonstrating high efficiency in introducing indel mutations in plant cells, comparable to or exceeding traditional plasmid-based methods, and allows for the regeneration of plants with desired genetic modifications.
Implementation Method 1
CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are loci containing multiple short direct repeats and are found in 40% of the sequenced bacteria and 90% of sequenced archaea. The Cas9 protein (or protein with similar function) is an important component of the typeII CRISPR/Cas system and forms an endonuclease, when combined with the crRNA and a second RNA termed the trans-activating cRNA (tracrRNA), which targets the invading pathogen DNA for degradation by the introduction of DNA double strand breaks (DSBs) at the position in the genome defined by the crRNA.
Implementation Method 2
A method involving an aqueous medium comprising a CRISPR-associated protein (Cas protein) and a CRISPR-CAS system guide RNA, with polyethylene glycol (PEG) and minimal or no glycerol, specifically optimized for plant cells
Implementation Method 3
Once a DNA DSB has been produced the cellular DNA repair machinery, particularly proteins belonging to the non-homologous end joining pathway, are involved in the re-ligation of the DNA ends. This process can lead to the loss or gain of a few nucleotides at the break, creating an INDEL mutation in the genomic DNA.
Data Source
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AI summary
Disclosed is a new method of providing plant cells with a targeted alteration in a DNA molecule. The method comprises contacting a population of plant cells comprising a DNA molecule, the DNA molecule having a target sequence, with an aqueous medium, wherein the aqueous medium comprises a CRISPR associated protein (CAS protein) or a CAS-like protein, and a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and wherein the aqueous medium comprises polyethylene glycol (PEG), but needs to be substantially free of glycerol.