Engineered Cross-Type Nucleic Acid Targeting for CRISPR Systems
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Solution Overview
Problem
Current CRISPR-Cas systems, particularly Type II systems, require a tracrRNA for crRNA maturation and DNA target sequence cleavage, which can be complex and less efficient compared to Type V systems that do not require tracrRNA, and there is a need for a more versatile system that can target multiple DNA sequences effectively.
Innovation Solution
Development of engineered CRISPR Class 2 cross-type-nucleic acid targeting nucleic acids (CRISPR Class 2 cross-type-NATNA) that combine Cpf1 and Cas9 nucleic acids, allowing for simultaneous binding and cleavage of multiple DNA sequences using a single complex, with the Cpf1-NATNA forming a complex with the Cpf1 protein and the Cas9-NATNA forming a complex with the Cas9 protein, enabling targeted DNA binding and cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Type II CRISPR-Cas systems are used, then DNA target sequence cleavage can be achieved, but the system requires tracrRNA which increases complexity and reduces efficiency
Solution Approach 1:
The patent extracts and removes the tracrRNA component from the CRISPR-Cas system, utilizing only the crRNA and Cas9 proteins to achieve DNA target sequence cleavage. This simplification eliminates the complexity introduced by tracrRNA while preserving the essential cleavage function through direct crRNA-guided Cas9 activity.
Solution Approach 2:
The engineered crRNA is designed to perform multiple functions: guiding Cas9 to target sequences and facilitating cleavage without requiring separate tracrRNA. The system achieves multi-functionality by making the crRNA itself sufficient for both targeting and cleavage induction, thereby reducing overall system complexity.
2Adaptability or versatility
If a single CRISPR-Cas system is used, then genome editing can be performed, but the ability to target multiple DNA sequences simultaneously is limited
Solution Approach 1:
The patent merges multiple CRISPR-Cas systems into a single integrated system where different crRNA molecules, each guided by distinct PAM sequences, work together within one cellular environment. This combination enables simultaneous targeting of multiple DNA sequences using a unified system architecture rather than separate systems.
Solution Approach 2:
The engineered CRISPR-Cas system is designed with universal capability to recognize and target multiple different DNA sequences through different crRNA-PAM combinations. This multi-functional design allows a single system to perform multiple targeting functions that would traditionally require separate specialized systems.
3Device complexity
If Type V CRISPR-Cas systems are used, then crRNA maturation is simplified without tracrRNA, but the ability to cleave DNA target sequences is reduced
Solution Approach 1:
The patent merges the advantages of Type V systems (simplified crRNA maturation without tracrRNA) with the DNA cleavage capability of Type II systems by using engineered Cas9 proteins that can function with crRNA alone. This combination achieves both simplified maturation and reliable cleavage in a single system.
Solution Approach 2:
The system uses composite functional elements where engineered Cas9 proteins combine the simplicity of Type V crRNA processing with the potent DNA cleavage activity needed for reliable targeting. This composite approach integrates the best features of both system types.
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 enhances the efficiency and versatility of genome editing by allowing for simultaneous binding and cleavage of multiple DNA sequences, improving the precision and efficacy of CRISPR-Cas systems by integrating the advantages of Type II and Type V systems into a single platform.
Implementation Method 1
the Cpf1-NATNA forming a complex with the Cpf1 protein
Implementation Method 2
the Cas9-NATNA forming a complex with the Cas9 protein
Implementation Method 3
DNA target sequence cleavage
Data Source
AI summary
The present disclosure provides engineered cross-type-nucleic-acid targeting nucleic acids and compositions thereof. Nucleic acid sequences encoding the engineered cross-type-nucleic-acid targeting nucleic acids, as well as expression cassettes, vectors and cells comprising such nucleic acid sequences, are described. Also, methods are disclosed for making and using the engineered cross-type-nucleic-acid targeting nucleic acids and compositions thereof.