Plant-Optimized Cpf1 Constructs for Precise Genome Editing
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Solution Overview
Problem
There is a need for more effective Cpf1-based genome editing technologies in plants, as existing methods have limitations in efficiency and specificity.
Innovation Solution
The use of plant-optimized recombinant nucleic acids encoding Cpf1, including nuclear localization signals, promoters, and guide RNAs, to enhance genome editing in plant cells through targeted sequence modification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional Cpf1-based genome editing methods are used in plants, then genome editing can be achieved, but the efficiency and mutation rates are limited
Solution Approach 1:
The patent applies parameter changes by optimizing the Cpf1 coding sequence for plant-specific codon usage patterns, adjusting the nucleotide composition and sequence parameters to match plant cellular environments. This optimization transforms the originally bacterial Cpf1 sequence into a version that is more efficiently transcribed and translated in plant cells, directly resolving the contradiction between achieving genome editing and maintaining consistent, reliable mutation rates across different plant systems
Solution Approach 2:
The patent implements local quality by strategically positioning nuclear localization signals (NLS) at specific locations within the Cpf1 protein structure. Rather than uniformly modifying the entire protein, the NLS sequences are placed at precise local positions (N-terminal and C-terminal regions) to ensure efficient nuclear targeting. This localized modification approach enhances the reliability of genome editing by ensuring the Cpf1 protein reaches its functional destination consistently, while maintaining overall protein functionality
2Reliability
If Cpf1 is introduced into plant cells without optimization, then genome editing function is achieved, but nuclear localization and expression efficiency are insufficient
Solution Approach 1:
The patent introduces nuclear localization signals (NLS) as intermediary elements that mediate the transport of Cpf1 protein from the cytoplasm to the nucleus. These NLS sequences act as molecular intermediaries that recognize and facilitate interaction with the nuclear pore complex, enabling efficient nuclear import. This intermediary mechanism resolves the contradiction by providing a dedicated transport pathway that simultaneously ensures reliable nuclear localization and maintains high protein expression levels
Solution Approach 2:
The patent applies preliminary action by pre-attaching nuclear localization signals to the Cpf1 protein sequence before the protein is expressed in plant cells. This preliminary modification ensures that as soon as the protein is synthesized, it is immediately directed to the nucleus rather than remaining in the cytoplasm. This preemptive localization strategy resolves the contradiction between nuclear localization efficiency and expression levels by ensuring the protein is correctly targeted from the moment of synthesis
3Manufacturing precision
If standard Cpf1 sequences are used, then genome editing capability is provided, but specificity and target site precision are insufficient
Solution Approach 1:
The patent applies segmentation by dividing the gRNA construct into distinct functional segments: a promoter region, a spacer sequence complementary to the target DNA, and a scaffold region. This segmented architecture allows each component to be independently optimized for its specific function. The spacer sequence is precisely designed to match the target site, while the scaffold maintains structural integrity and Cpf1 binding. This segmentation resolves the contradiction between editing precision and construct complexity by organizing the nucleic acid into modular, functionally-defined segments that can be systematically designed and assembled
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
The described methods achieve higher mutation rates and specificity in genome editing, enabling targeted modifications in various plant species, including maize, soybean, and tobacco, with efficiencies up to 64% mutation frequency at target sites.
Implementation Method 1
the guide polynucleotide and a Cpf1 endonuclease encoded by the recombinant nucleic acid are capable of forming a complex that can recognize, bind to, and optionally nick or cleave the target sequence
Data Source
AI summary
This disclosure is related to plant-optimized recombinant nucleic acids encoding Cpf1 and their use in planta. Also disclosed are compositions, expression cassettes, and plant cells comprising the recombinant nucleic acids as well as methods and kits for modifying a target sequence in a plant genome using the recombinant nucleic acids.