Cpf1 Nuclease Targeted DNA Alteration in Plant Cells
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Solution Overview
Problem
Current methods for targeted DNA alteration in plant cells are not always successful or efficient, and existing methodologies optimized for animal cells are not well-suited for plant cells, leading to a need for reliable, efficient, and targeted methods specifically designed for plant cells.
Innovation Solution
A method for targeted alteration of duplex DNA in plant cells using the Cpf1 protein and a crRNA guide sequence, where the Cpf1 protein is expressed transiently in plant protoplasts, along with the crRNA, to introduce targeted double-strand breaks at specific genomic loci, allowing for precise modification of the plant genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If exogenous DNA fragments are added to the genome of a cell, then new properties are conferred to the cell, but the insertion positions are not controlled and precision is reduced
Solution Approach 1:
The system is divided into three functional components: a guide RNA that recognizes the target sequence, a Cas9 nuclease that creates the break, and a donor DNA template that provides the repair sequence. This segmentation allows each component to be optimized independently for its specific function, achieving both versatility in target selection and precision in modification location.
Solution Approach 2:
The guide RNA acts as an intermediary that bridges the target genome sequence and the Cas9 nuclease. It contains a spacer sequence complementary to the target, allowing specific recognition and binding, thereby directing the nuclease to the exact location where modification is desired, solving the precision problem.
2Manufacturing precision
If CRISPR-Cas9 system is used for targeted DNA alteration, then precise modification is achieved, but the efficiency and success rate in plant cells are reduced
Solution Approach 1:
The system uses a protoplast transformation approach with optimized parameters including PEG-mediated delivery, specific incubation times and temperatures, and controlled regeneration conditions. These parameter optimizations significantly improve the efficiency of DNA delivery and expression in plant cells while maintaining precision.
Solution Approach 2:
The method performs preliminary optimization by selecting highly efficient guide RNA sequences with specific characteristics (length, composition, secondary structure) before actual transformation. This preliminary selection ensures high efficiency during the actual mutagenesis process while maintaining target precision.
3Adaptability or versatility
If traditional genome editing methods are used, then broad applicability is achieved, but reliability in plant cells is reduced
Solution Approach 1:
The CRISPR-Cas9 system provides universality through the modular guide RNA design that can be programmed to recognize any DNA sequence with appropriate PAM sites. The same core machinery (Cas9 nuclease) can target different loci by simply changing the guide RNA, achieving broad applicability across different plant species and genes while maintaining high reliability through optimized delivery methods.
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 method enables efficient and precise introduction of targeted alterations, such as insertions, deletions, or modifications of base pairs, in plant cells, with higher mutagenesis frequencies compared to existing CRISPR-Cas9 systems, facilitating the regeneration of modified plants with improved traits.
Implementation Method 1
a crRNA comprising a guide sequence for targeting said Cpf1 protein to the site of the duplex DNA comprising the target sequence
Implementation Method 2
exposing said duplex DNA in said cells or protoplasts to: a Cpf1 protein; and a crRNA comprising a guide sequence for targeting said Cpf1 protein to the site of the duplex DNA comprising the target sequence
Data Source
AI summary
The current invention relates to methods of targeted genetic alteration in plant cells, as well as to plant cells and plants thus obtained. In the method, a Cpf1 protein and a crRNA is employed to provide for targeted alteration, in particular increased biallelic alteration, of a DNA duplex with increased efficacy.