Engineered Cpf1 Direct Repeat Variants for Multi-Target Genome Editing
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Solution Overview
Problem
Current genome editing technologies, such as CRISPR-Cas systems, face challenges in efficiently targeting multiple positions within the eukaryotic genome while minimizing off-target effects and genetic variation in patient populations.
Innovation Solution
Engineered Cpf1 polynucleotides are designed to direct the activity of a CRISPR protein to multiple targets using a single crRNA, incorporating direct repeats with mutations to enhance specificity and efficacy, and are delivered using novel viral and lipid particles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If a single CRISPR guide RNA is used to target multiple positions in the genome, then the complexity of the system is reduced and ease of operation is improved, but the precision of targeting and manufacturing precision deteriorates due to increased off-target effects
Solution Approach 1:
The patent segments the direct repeat sequence into multiple variants (DR1, DR2, DR3, etc.), each with specific mutations. This segmentation allows the guide RNA to maintain multiple targeting functions while reducing off-target effects, as each DR variant contributes to the specificity at different target positions.
Solution Approach 2:
The patent applies local quality by introducing specific mutations at different positions within the direct repeat sequence. Each mutation (e.g., at positions 3, 6, 9, 12, 15, 18, 21, 24, 27, 30) is strategically placed to enhance binding affinity or specificity at particular target sites, allowing differential optimization of each target position within the same guide RNA.
2Manufacturing precision
If direct repeats with mutations are introduced to enhance specificity, then manufacturing precision and targeting accuracy are improved, but the device complexity and difficulty of manufacture increase
Solution Approach 1:
The patent systematically changes parameters of the direct repeat sequence by introducing mutations at specific positions (3, 6, 9, 12, 15, 18, 21, 24, 27, 30). These parameter changes involve substituting nucleotides to create DR variants with optimized binding properties, thereby enhancing targeting precision through controlled sequence modifications.
3Reliability
If multiple direct repeat variants are incorporated into a single guide RNA to reduce off-target effects, then reliability and safety are improved, but the quantity of substance and molecular weight increase
Solution Approach 1:
The patent creates a universal guide RNA structure that can target multiple positions in the genome simultaneously. By incorporating multiple DR variants (DR1, DR2, DR3, etc.) within a single guide RNA molecule, the system achieves multi-functionality, allowing one guide RNA to perform the work of multiple guides while maintaining high specificity through the mutated DR sequences.
Data Source
AI summary
The present invention generally relates to systems, methods and compositions related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof. Provided herein are engineered Cpf1 polynucleotides that can direct the activity of a CRISPR protein to multiple targets using a single crRNA. The guide sequences can target a sequence in a eukaryotic cell, for example, an animal or a plant cell. The animal cell can be a human or a nonhuman cell. Additionally, the present invention relates to methods for developing or designing CRISPR-Cas system-based therapy or therapeutics.