XNA-gRNA CRISPR Guide RNA Stability and Specificity
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Solution Overview
Problem
Current CRISPR-Cas9 systems face inefficiencies in genome editing and gene regulation, particularly in primary cells, due to off-target effects, stability issues, and limited transfectability, which hinder precise genome editing and gene expression modulation.
Innovation Solution
The development of synthetic chimeric xenonucleic acid guide RNA (XNA-gRNA) constructs with a neutral non-phosphate backbone, resistant to nucleases and enhanced binding affinity, used in conjunction with chemically synthesized trans-activating CRISPR RNA to recruit Cas9 nuclease for targeted gene editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If native bacterial crRNA-tracrRNA duplex is used, then CRISPR system shows good genome editing capability, but the structure is complex and transfectability is limited
Solution Approach 1:
The patent combines crRNA and tracrRNA into a single guide RNA (sgRNA) molecule, simplifying the overall structure while maintaining CRISPR functionality. This merging reduces the complexity of delivering multiple RNA components separately and improves transfectability by using a single RNA construct that retains genome editing capability.
Solution Approach 2:
The patent employs chemically modified nucleosides and nucleotides to create composite RNA structures with enhanced properties. These modifications include phosphorothioate backbone linkages and 2'-O-methyl modifications, which improve stability and transfectability while maintaining the guide RNA's ability to direct Cas9 to target sequences.
2Ease of manufacture
If standard RNA guide sequences are used, then CRISPR system is easy to implement, but off-target effects occur and stability is reduced
Solution Approach 1:
The patent modifies the chemical parameters of the guide RNA by incorporating phosphorothioate backbone linkages and 2'-O-methyl nucleoside modifications. These parameter changes enhance the thermal stability and nuclease resistance of the RNA, reducing degradation and off-target effects while maintaining ease of synthesis through standard oligonucleotide synthesis methods.
Solution Approach 2:
The patent uses chemically synthesized oligonucleotides with modified backbones that are more stable and resistant to degradation. These modified guide RNAs maintain high specificity for target sequences while being easier to manufacture with consistent quality, reducing off-target effects through improved molecular stability.
3Productivity
If CRISPR-Cas9 is used for genome editing, then gene knockout efficiency is improved, but off-target effects and stability issues persist
Solution Approach 1:
The patent introduces chemically modified nucleosides and phosphorothioate linkages as intermediaries between the guide RNA and target DNA. These modifications act as protective intermediaries that enhance binding specificity and reduce off-target effects by improving the precision of RNA-DNA hybridization while maintaining high gene knockout efficiency.
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 significantly enhances the efficiency and specificity of CRISPR-mediated genome editing, reducing off-target effects and improving stability, allowing for precise gene regulation and editing in various cell types, including primary cells and human cells.
Implementation Method 1
synthetic chimeric xenonucleic acid guide RNA (XNA-gRNA) constructs... used in conjunction with chemically synthesized trans-activating CRISPR RNA to recruit Cas9 nuclease
Implementation Method 2
XNA-gRNA constructs with a neutral non-phosphate backbone, resistant to nucleases
Implementation Method 3
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a method of gene editing that utilizes the Cas9 protein and specific guide RNAs to either disrupt host genes or insert sequences of interest
Data Source
AI summary
The invention provides specific synthetic chimeric xenonucleic acid guide RNA; s(XNA-gRNA) for enhancing crispr mediated genome editing efficiency. The invention also provides methods and compositions for inducing CRISPR/Cas-based gene editing/regulation (e.g., genome editing or gene expression) of a target nucleic acid (e.g., target DNA or target RNA) in a cell. The methods include using single guide RNAs (sgRNAs) that have been chemically modified with xeno nucleic acids which enhance gene regulation of the target nucleic acid in a primary cell for use in ex vivo therapy or in a cell in a subject for use in in vivo therapy. Additionally, provided herein are methods for preventing or treating a genetic disease in a subject by administering a sufficient amount of a sgRNA that has been chemically modified with xeno nucleic acids to correct a mutation in a target gene associated with the genetic disease.