CRISPR-Cas9 Multi-sgRNA Targeting for Selective, Specific Cell Killing
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Solution Overview
Problem
CRISPR-Cas9 systems face challenges with off-target activity and toxicity, leading to unintended genetic alterations such as whole chromosome arm loss, limiting their effectiveness in treating diseases associated with somatic mutations like cancer.
Innovation Solution
A method to identify somatic mutations in tumors that produce protospacer adjacent motifs (PAMs) and design a CRISPR-Cas9 system with sgRNAs targeting these sites, including multi-target sgRNAs to induce multiple double-strand breaks (DSBs) in non-coding regions, thereby selectively killing cancer cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas9 system is used to target and cut DNA sequences, then gene editing capability is improved, but off-target activity and toxicity increase leading to unintended genetic alterations
Solution Approach 1:
The patent divides the single sgRNA targeting function into multiple parallel sgRNAs, each targeting a specific somatic mutation. This segmentation allows the system to focus cutting activity only on mutant alleles with high specificity, avoiding off-target effects on wild-type sequences while maintaining effective gene editing capability.
Solution Approach 2:
The patent applies local quality by designing sgRNAs that are specific to the unique sequence context created by somatic mutations. Each sgRNA is tailored to recognize and cut only at the mutated site, ensuring that the editing action is localized precisely to the harmful mutation without affecting other genomic regions.
2Productivity
If CRISPR-Cas9 system targets multiple mutations simultaneously, then cell killing efficiency is improved, but system complexity increases
Solution Approach 1:
The patent implements universality by using a single Cas9 enzyme that can perform the same cutting function across multiple different target sites. By combining multiple sgRNAs that guide the universal Cas9 to different somatic mutations, the system achieves multi-functional targeting capability, simultaneously inducing multiple DSBs to enhance cell killing efficiency without requiring multiple different enzymatic systems.
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 approach achieves selective cell killing with high specificity and efficiency, inducing genomic instability and polyploidization, ultimately leading to cancer cell death while minimizing off-target effects.
Implementation Method 1
the CRISPR-Cas9 system has been rapidly adopted by scientists as the tool of choice for gene editing (5-7). CRISPR-Cas9 works by introducing a double-strand break (DSB) as directed by a complementary single-guide RNA (sgRNA) sequence in the presence of a protospacter adjacent motif (PAM)
Implementation Method 2
performing next generation sequencing of DNA obtained from the tumor sample and the normal sample to produce a tumor sequence and a normal sequence
Data Source
AI summary
A CRISPR-Cas9 system for treating a disease, disorder, or condition associated with one or more somatic mutations in a subject in need of treatment thereof is disclosed. The system comprises a sgRNA-guided Cas9, wherein the sgRNA targets between about 1 to about 50 mutations in a target cell. The CRISPR-Cas9 system can be used to treat diseases, disorders, or conditions associated with one or more somatic mutations, including cancers, autoimmune diseases, and/or neurodegenerative diseases. Additionally, the present disclosure relates to methods of identifying somatic mutations in a tumor that produce a protospacer adjacent motif (PAM) and methods of designing a CRISPR-Cas 9 system to target PAMs identified in a tumor sample obtained from a subject.