CRISPR/Cas Ortholog Engineering for Scalable Genome Targeting
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Solution Overview
Problem
Existing genome-editing technologies are costly, complex, and limited in their ability to target multiple positions within the eukaryotic genome, necessitating the development of affordable, scalable, and versatile systems for precise genome perturbation.
Innovation Solution
The CRISPR/Cas system utilizes a single Cas enzyme programmed by a short RNA molecule for specific DNA targeting, enabling efficient genome editing with a CRISPR complex comprising Cas orthologs and optimized guide sequences, and includes chimeric Cas9 proteins and engineered compositions for enhanced specificity and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional genome-editing technologies (designer zinc fingers, TALEs, homing meganucleases) are used, then targeted genome perturbation can be achieved, but the systems are costly, complex, and limited in scalability
Solution Approach 1:
The CRISPR-Cas9 system employs a universal Cas9 enzyme that can target multiple genomic locations through different guide RNAs, replacing the need for multiple customized protein systems. This multi-functional approach allows a single enzyme to perform targeted genome perturbation at various sites, reducing overall system complexity while maintaining reliability.
Solution Approach 2:
The system uses RNA molecules as programmable guides that can be easily synthesized and modified to target different sequences. Instead of creating new proteins for each target, the system copies the targeting function through programmable RNA sequences, significantly simplifying the engineering process and reducing complexity.
2Measurement precision
If traditional genome-editing technologies are used, then specific DNA targeting is possible, but the methodology is costly and not easily scalable
Solution Approach 1:
The system replaces expensive, complex protein engineering with inexpensive, easily synthesized RNA molecules. These guide RNAs can be rapidly produced and modified at low cost, making the system highly scalable and accessible without sacrificing targeting specificity.
Solution Approach 2:
The system achieves different targeting specificities by changing the nucleotide sequence parameters of the guide RNA rather than modifying protein structures. This parameter change approach allows easy optimization of targeting precision while maintaining cost-effectiveness and scalability.
3Reliability
If traditional genome-editing technologies are used, then genome perturbation can be achieved, but the ability to target multiple positions is limited
Solution Approach 1:
A single Cas9 enzyme can be programmed with different guide RNAs to target multiple positions in the genome, providing versatile multi-position editing capability. This universal platform maintains genome perturbation effectiveness while enabling simultaneous or sequential targeting of multiple loci, greatly enhancing adaptability.
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 methodologies, allowing for precise and widespread applications in gene regulation, therapy, and disease diagnosis by providing robust and versatile tools for modifying target polynucleotides across various cell types.
Implementation Method 1
a guide sequence that is hybridized to a target sequence
Implementation Method 2
the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence
Implementation Method 3
the tracr mate sequence that is hybridized to the tracr sequence
Data Source
AI summary
The invention provides for engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are compositions and methods related to components of a CRISPR complex particularly comprising a Cas ortholog enzyme.