CRISPR-Cas System Scalable Genome Editing via Programmable RNA
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Solution Overview
Problem
Current genome editing technologies, such as CRISPR-Cas systems, require customized proteins for specific sequence targeting, which can be complex and costly, and lack scalability for targeting multiple positions within eukaryotic genomes.
Innovation Solution
The development of a CRISPR-Cas system that uses a single Cas enzyme programmed by a short RNA molecule to recognize specific DNA targets, with modified guide RNAs and enzymes having reduced nuclease activity, allowing for efficient and scalable genome editing by recruiting adaptor proteins with functional domains.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If customized proteins are used for specific sequence targeting, then targeting precision is improved, but device complexity and manufacturing cost increase
Solution Approach 1:
The CRISPR-Cas system employs a universal Cas enzyme that can be programmed to target different DNA sequences through interchangeable guide RNAs, eliminating the need for customized proteins for each target site. This single enzyme system performs multiple targeting functions, resolving the contradiction between targeting precision and system complexity
Solution Approach 2:
The system uses RNA molecules as programmable guides that can be easily synthesized and modified to match different target sequences. These guide RNAs serve as information carriers that direct the Cas enzyme to specific genomic locations, replacing complex customized protein designs with simpler RNA templates
2Measurement precision
If customized proteins are used for each target, then targeting specificity is improved, but productivity and scalability deteriorate
Solution Approach 1:
A single CRISPR-Cas system with programmable guide RNAs can simultaneously or sequentially target multiple genomic positions, enabling high-throughput functional screening across hundreds or thousands of genes without requiring multiple customized protein systems, thus improving both specificity and productivity
Solution Approach 2:
The system allows rapid reprogramming by changing the guide RNA sequence parameters to match different target sites. This parameter-based reconfiguration enables scalable targeting of multiple positions using the same Cas enzyme, eliminating the need to generate new proteins for each target
3Object-affected harmful factors
If nuclease activity is reduced in CRISPR enzyme, then safety and control are improved, but DNA cleavage efficiency decreases
Solution Approach 1:
The invention separates the DNA cleavage function from the genome targeting and verification functions. By removing or reducing nuclease activity, the system eliminates harmful off-target cleavage effects while maintaining the ability to precisely locate and identify target sites through guide RNA-directed binding
Solution Approach 2:
The system introduces intermediary molecules or mechanisms that facilitate DNA modification without requiring direct nuclease cleavage. This intermediary approach allows for controlled genome editing while reducing the harmful effects associated with high nuclease activity, balancing safety and efficiency
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 simplifies genome editing by reducing the need for customized proteins, enhances scalability, and enables precise targeting of multiple genomic locations, facilitating the cataloging of genetic factors associated with biological functions and diseases.
Implementation Method 1
a guide RNA (sgRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest
Data Source
AI summary
The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. Provided are structural information on the Cas protein of the CRISPR-Cas system, use of this information in generating modified components of the CRISPR complex, vectors and vector systems which encode one or more components or modified components of a CRISPR complex, as well as methods for the design and use of such vectors and components. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for utilizing the CRISPR-Cas system. In particular the present invention comprehends optimized functional CRISPR-Cas enzyme systems.