Segmented CRISPR-Cas9 Nickase System for Specific Genome Editing
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Solution Overview
Problem
Current genome-editing technologies, particularly CRISPR-Cas systems, face challenges with off-target modifications, limiting their translation into clinical therapies due to lack of specificity and precision, especially in dividing cells and RNA-guided systems.
Innovation Solution
Development of non-Class I engineered CRISPR-Cas polynucleotide targeting systems comprising multiple Cas proteins or a Cas protein with non-Cas proteins, featuring guide molecules for site-specific binding and allosteric interactions to enhance specificity and control over catalytic events.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR-Cas systems are used for genome editing, then editing efficiency and speed are improved, but off-target modifications increase reducing specificity
Solution Approach 1:
The patent divides the single Cas9 protein into two separate Cas proteins (Cas9a and Cas9b), each capable of binding to the target sequence but unable to perform full cleavage alone. This segmentation requires two separate binding events to achieve the desired editing outcome, thereby increasing specificity while maintaining editing efficiency.
Solution Approach 2:
The patent introduces a bridge helix domain that acts as an intermediary structural element facilitating allosteric communication between the two Cas9 proteins. This intermediary mechanism ensures that both proteins must be correctly positioned and bound to the target for catalytic activity, reducing off-target effects.
2Ease of operation
If DNA guided systems are used for base editing, then ease of implementation is improved, but inherent off-target editing occurs during cell replication
Solution Approach 1:
The patent segments the DNA-guided editing system into two separate DNA-guided Cas9 proteins that must simultaneously bind to the target site. This dual-binding requirement creates a higher threshold for on-target activity while significantly reducing the probability of off-target editing during cell replication.
3Manufacturing precision
If RNA guided CRISPR-Cas systems are used, then precision of targeting is improved, but off-target base editing still occurs
Solution Approach 1:
The patent applies segmentation to the RNA-guided system by using two separate RNA-guided Cas9 proteins instead of one. Each RNA guide must find and bind its complementary sequence, and both proteins must be present and correctly positioned for editing to occur, thereby maintaining precision while reducing off-target effects.
Solution Approach 2:
The bridge helix domain serves as an intermediary that transmits allosteric signals between the two RNA-guided Cas9 proteins, ensuring that both are correctly bound to the target sequence before catalytic activity is permitted, thus reducing off-target editing.
4Manufacturing precision
If multiple Cas proteins are used in the system, then specificity and control are improved, but system complexity increases
Solution Approach 1:
The patent merges the functionality of two Cas9 proteins into a single functional unit where both proteins work cooperatively at the target site. The bridge helix domain facilitates this merging by enabling allosteric communication, allowing the system to achieve high specificity while managing complexity through functional integration.
Solution Approach 2:
Each Cas9 protein in the patent is designed to be multifunctional, capable of binding to the target sequence, interacting with the bridge helix domain, and participating in the catalytic complex. This universality reduces the need for additional specialized components, managing system complexity while maintaining high specificity.
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
The system increases specificity and reduces off-target events, improving the safety and efficacy of CRISPR-Cas systems for therapeutic applications by allowing precise targeting and modification of nucleic acids.
Implementation Method 1
a guide molecule capable of forming a complex with at least one of the two or more Cas proteins and directing site-specific binding to a target sequence of a target polynucleotide
Implementation Method 2
the first Cas protein and the second Cas protein allosterically interact upon target recognition to coordinate nicking of the first and second strands of the dsDNA polynucleotide
Data Source
AI summary
Described herein are non-Class I engineered CRISPR-Cas systems and components thereof, formulations thereof, cells thereof, and organisms thereof. Methods of making and using the CRISPR-Cas system described herein.