Cas12F Nuclease Single Guide RNA Deletion
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Solution Overview
Problem
Existing CRISPR/Cas systems require multiple guide RNAs to achieve efficient deletions in DNA regulatory sequences, which can be problematic due to varying cutting efficiencies and the need for simultaneous cuts at different sites.
Innovation Solution
The use of Cas12F nucleases, such as Cas12f.4, Cas12f.5, and Cas12f.6, which can induce deletions in target sites with a single guide RNA, allowing for longer deletions and reducing the need for multiple guide RNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple guide RNAs are used to achieve efficient deletions in DNA regulatory sequences, then deletion efficiency is improved, but system complexity increases due to the need for multiple components and varying cutting efficiencies
Solution Approach 1:
The patent segments the guide RNA system into a single multifunctional guide RNA that can direct Cas12F to multiple target sites within DNA regulatory sequences. This single guide RNA contains spacer sequences that enable it to recruit Cas12F to multiple locations, eliminating the need for multiple separate guide RNAs while maintaining high deletion efficiency in promoter regions and other regulatory elements.
Solution Approach 2:
The single guide RNA is designed with universal functionality to perform multiple cutting tasks. It can simultaneously or sequentially direct Cas12F to multiple target sites within the same regulatory sequence, making one guide RNA component sufficient for achieving complex deletion patterns that previously required multiple guide RNAs.
2Manufacturing precision
If multiple guide RNAs are used to target different sites in DNA regulatory sequences, then comprehensive editing coverage is improved, but operational difficulty increases due to the need for simultaneous cuts
Solution Approach 1:
The guide RNA is segmented to include multiple spacer sequences, each targeting a specific site within the DNA regulatory sequence. This segmentation allows the single guide RNA to cover multiple editing locations without requiring multiple separate guide RNA components, simplifying the operational coordination while maintaining comprehensive editing coverage.
Solution Approach 2:
The guide RNA is pre-designed with all necessary spacer sequences incorporated into a single molecular structure. This preliminary configuration ensures that all target sites are covered before the editing process begins, eliminating the need for complex real-time coordination of multiple guide RNAs during the cutting process.
3Device complexity
If Cas12F nuclease is used with a single guide RNA to induce deletions, then system simplicity is improved, but deletion length may be limited compared to multiple guide RNA approaches
Solution Approach 1:
The Cas12F nuclease system with a single guide RNA exhibits dynamic cutting behavior, where the nuclease can perform sequential cuts at multiple sites within the regulatory sequence after being recruited by the single guide RNA. This dynamic operation allows the system to achieve long deletion lengths comparable to multiple guide RNA approaches, while maintaining system simplicity.
Solution Approach 2:
The single guide RNA enables continuous useful action by maintaining Cas12F recruitment and activity at multiple target sites within the regulatory sequence. The Cas12F nuclease continuously performs cutting operations along the DNA sequence guided by the single RNA molecule, achieving comprehensive deletions without interruption or the need for multiple separate guide RNA components.
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
Cas12F nucleases enable efficient and precise deletions in DNA regulatory sequences, such as promoter regions, in plant cells like soybean and corn, leading to altered gene expression and potential phenotypic changes without the need for multiple guide RNAs.
Implementation Method 1
The CRISPR/Cas Type V system is a type of CRISPR system that has a 5′-TTN motif and cuts target sequences with sticky ends, such as Cpf1, C2c1, CasX, and CasY.
Implementation Method 2
It recognizes the PAM motif of 3′-NGG and cuts the target sequence with blunt ends.
Data Source
AI summary
Methods for obtaining plant cells, plants, and plant parts, including soybean and corn plant cells, comprising Cas12F nucleases and at least one guide RNA that target the DNA regulatory sequences including a promoter region, thereby producing a gene allele with altered expression levels. The methods can be used to generate alleles of genes of interest by selecting for phenotypes conferred by the altered expression of the gene(s). Also provided are soybean plant cells and com plant cells comprising synthetic polynucleotides that provide for gene alleles with altered expression levels.