Allele-Specific CRISPR Editing of NRAS and KRAS Mutations
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CRISPR-Cas nucleases lack specificity in discriminating single-nucleotide mutations from wild-type alleles, making it challenging to effectively target and disrupt oncogenic mutations such as NRAS Q61L and KRAS G12D in cancer cells.
Innovation Solution
The use of highly specific Cas nuclease systems, such as those comprising CasPhi.12 or CasM.265466, in conjunction with guide nucleic acids that are complementary to target sequences of mutant alleles, allows for allele-specific editing of NRAS and KRAS genes, enabling selective cleavage of mutant alleles without affecting wild-type alleles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current CRISPR-Cas nucleases are used to target mutant alleles, then the editing capability is achieved, but the specificity to discriminate single-nucleotide mutations from wild-type alleles is insufficient
Solution Approach 1:
The patent introduces a mismatch tolerance mechanism where the guide nucleic acid is designed with specific mismatch capabilities at certain positions. The system allows mismatches at non-critical positions while maintaining strict matching requirements at critical positions, enabling discrimination of single-nucleotide mutations. This local quality differentiation in matching stringency resolves the contradiction by providing high specificity for mutant allele identification while maintaining reliable editing capability.
Solution Approach 2:
The patent employs modified nucleic acid bases with altered chemical properties (such as LNA, 2'-O-methyl RNA, or phosphorothioate modifications) to change the hybridization parameters of the guide nucleic acid. These parameter changes enhance the discrimination ability between mutant and wild-type alleles by creating differential binding affinities, thereby improving both specificity and reliability simultaneously.
2Manufacturing precision
If guide nucleic acid sequences are designed to match mutant alleles, then allele-specific editing is enabled, but the risk of off-target effects on wild-type alleles increases due to low specificity
Solution Approach 1:
The guide nucleic acid design implements local quality differentiation by applying different matching stringency requirements to different regions. Critical positions (where the mutation occurs) require perfect matching, while non-critical positions tolerate mismatches. This ensures high precision for mutant allele targeting while preventing off-target effects on wild-type alleles, as the modified bases create sufficiently different binding affinities to distinguish between mutant and wild-type sequences.
Solution Approach 2:
The patent uses modified nucleic acid bases as intermediary elements that mediate the recognition between the guide nucleic acid and target alleles. These modified bases act as chemical mediators that enhance the discrimination capability, allowing the system to achieve high allele-specific editing precision while minimizing off-target effects through differential hybridization stability.
3Measurement precision
If highly specific Cas nuclease systems are used, then selective cleavage of mutant alleles is achieved, but the system complexity increases compared to conventional CRISPR-Cas systems
Solution Approach 1:
The patent achieves high discrimination precision by changing the chemical parameters of the guide nucleic acid through modifications such as LNA, 2'-O-methyl RNA, or phosphorothioate linkages. These parameter changes enhance binding specificity without requiring complex protein engineering or multi-component systems, thereby maintaining relative system simplicity while achieving the desired precision.
Solution Approach 2:
The patent uses simplified Cas nuclease variants that retain core cutting functionality but have reduced complexity compared to wild-type Cas9. By copying only the essential functions and using chemically modified guide nucleic acids to provide the discrimination capability, the system achieves high precision with lower overall complexity.
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 achieves precise editing of mutant alleles, leading to reduced cell proliferation and induction of apoptosis in cancer cells, thereby providing a potential therapeutic strategy for treating disorders associated with NRAS and KRAS mutations.
Implementation Method 1
hybridization of the guide nucleic acid to the target sequence of the mutant allele
Implementation Method 2
the effector protein selectively cleaves the mutant allele
Data Source
AI summary
Provided herein are compositions, systems and methods for allele-specific editing of a target gene in a cell, comprising contacting the cell with an effector protein or a polynucleotide encoding the same, and a guide nucleic acid or a polynucleotide encoding the same. These effector proteins may be characterized as CRISPR-associated (Cas) proteins. Various compositions, systems, and methods of the present disclosure may leverage the activities of these effector proteins for the modification, detection, and engineering the NRAS or KRAS gene.