Cpf1 Nuclease Temperature Adaptation for Genomic Editing
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Solution Overview
Problem
Current genomic modification techniques, such as those using CRISPR-Cas9, often require optimal temperatures that may not align with the conditions needed for certain applications, limiting their effectiveness, especially in plant genomic editing where temperature-specific activities of enzymes like Cpf1 nucleases are suboptimal.
Innovation Solution
Development of Cpf1 CRISPR systems that maintain activity across a broad temperature range, allowing for site-specific genomic modifications by introducing DNA constructs encoding modified Cpf1 proteins with enhanced activity at lower temperatures, enabling precise editing of genomic DNA sequences and modulation of gene expression in various organisms, including plants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If Cpf1 nucleases are used for genomic modification, then site-specific editing capability is improved, but temperature range for optimal activity is limited
Solution Approach 1:
The patent applies parameter changes by modifying the Cpf1 nuclease protein structure through mutagenesis to alter its temperature sensitivity parameters. Specific amino acid substitutions are introduced to shift the optimal activity temperature range from high temperatures to lower temperatures, enabling the enzyme to function effectively across a broader temperature spectrum while maintaining its site-specific editing precision.
Solution Approach 2:
The invention creates dynamic adaptability by engineering Cpf1 variants that can adjust their activity across different temperature conditions. The modified nucleases exhibit enhanced thermal flexibility, allowing the same enzyme variant to maintain functionality both during tissue culture operations at lower temperatures and during the actual genomic editing process at higher temperatures.
2Power
If wild-type Cpf1 nucleases are used, then high-temperature activity is improved, but low-temperature application effectiveness deteriorates
Solution Approach 1:
The patent systematically changes the temperature activity parameters of Cpf1 nucleases through directed mutagenesis. By substituting specific amino acid residues, the enzyme's thermal response characteristics are modified to reduce temperature sensitivity, enabling reliable activity at lower temperatures used in plant tissue culture while preserving or enhancing high-temperature editing effectiveness.
3Manufacturing precision
If temperature-specific enzyme activity is optimized, then editing precision at optimal temperature is improved, but applicability to diverse temperature conditions deteriorates
Solution Approach 1:
The invention achieves universality by creating Cpf1 nuclease variants that can perform genomic editing functions across multiple temperature conditions. The modified enzymes serve dual purposes: maintaining high precision at elevated temperatures during editing while also being effective at lower temperatures during tissue culture operations, thereby eliminating the need for separate enzyme optimizations for different temperature regimes.
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
Enables efficient and precise modification of genomic DNA sequences and gene expression regulation across a wide temperature range, facilitating applications in plant research and biotechnology by ensuring consistent enzyme activity, even in suboptimal temperature conditions.
Implementation Method 1
CRISPR nucleases use a guide molecule, often a guide RNA molecule, that interacts with the nuclease and base pairs with the targeted DNA, allowing the nuclease to produce a double-stranded break (DSB) at the desired site
Data Source
AI summary
Compositions and methods for modifying genomic DNA sequences are provided. The methods produce double-stranded breaks (DSBs) at pre-determined target sites in a targeted DNA sequence, resulting in mutation, insertion, and/or deletion of DNA sequences at the targeted site(s). Compositions comprise DNA constructs comprising nucleotide sequences that encode a Cpf1 protein operably linked to a promoter that is operable in the cells of interest. The DNA constructs can be used to direct the modification of genomic DNA at pre-determined locations. Methods to use these DNA constructs to modify genomic DNA sequences are described herein. Additionally, compositions and methods for modulating the expression of genes are provided. Compositions comprise DNA constructs comprising a promoter that is operable in the cells of interest operably linked to nucleotide sequences that encode a mutated Cpf1 protein with an abolished ability to produce DSBs, optionally linked to a domain that regulates transcriptional activity. The methods can be used to up- or down-regulate the expression of genes at predetermined genomic loci.