CRISPR-Cas Guide RNA Design for Scalable Nucleotide Repeat Editing
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Solution Overview
Problem
Current genome-editing techniques are not affordable, easy to set up, and scalable for targeting multiple positions within the eukaryotic genome, limiting their effectiveness in treating nucleotide repeat disorders.
Innovation Solution
Development of a self-inactivating CRISPR-Cas system comprising regulatory elements and guide sequences arranged in a 5' to 3' orientation, which includes a CRISPR enzyme complexed with guide and tracr mate sequences, allowing for sequence-specific binding and self-inactivation to limit activity duration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If current genome-editing techniques (designer zinc fingers, TALEs, homing meganucleases) are used to target multiple positions in the eukaryotic genome, then genome editing capability is achieved, but the techniques are not affordable, easy to set up, and scalable
Solution Approach 1:
The CRISPR-Cas9 system employs a universal guide RNA mechanism that can direct the Cas9 enzyme to multiple different genomic positions by simply changing the guide sequence, enabling one system to perform multiple targeting functions without requiring different enzyme complexes for each target site
Solution Approach 2:
The system divides the genome editing function into two separate components: the Cas9 enzyme and the guide RNA. This segmentation allows the guide RNA to be easily modified to target different positions while the Cas9 enzyme remains constant, improving ease of setup and scalability for multiple targets
2Productivity
If CRISPR-Cas system activity is maintained continuously, then genome editing efficiency is improved, but off-target activity increases
Solution Approach 1:
The system uses transient expression of the CRISPR-Cas9 components, where the guide RNA and Cas9 are expressed temporarily to perform genome editing, then naturally degrade or are degraded, limiting the duration of activity to reduce off-target effects while maintaining sufficient editing efficiency during the active period
Solution Approach 2:
The system employs control mechanisms such as inducible promoters or regulated expression systems that prevent CRISPR-Cas9 activity until the appropriate time, allowing preparation and targeting planning without unintended off-target editing events
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 CRISPR-Cas system effectively targets and edits nucleotide repeat sequences, reducing off-target activity and enabling precise genome engineering for treating disorders like Fragile X syndrome and Huntington's disease.
Implementation Method 1
a first guide sequence capable of hybridizing to a target DNA
Implementation Method 2
the CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that can be hybridized or can be hybridizable to the target sequence
Data Source
AI summary
The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences especially for use as to nucleotide repeat disorders. Provided are delivery systems and tissues or organ which are targeted as sites for delivery especially for use as to nucleotide repeat disorders. Also provided are vectors and vector systems some of which encode one or more components of a CRISPR complex or system especially for use as to nucleotide repeat disorders, as well as methods for the design and of such. Also provided are methods of directing CRISPR complex or system formation in eukaryotic cells especially for use as to nucleotide repeat disorders including with consideration of specificity for target recognition and avoidance of toxicity and editing or modifying a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.