Guide Nucleic Acid Targeting for Safe Exogenous DNA Integration
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Solution Overview
Problem
Current methods for expressing exogenous genes in cells often result in random integration, disrupting native gene expression and potentially leading to malignant transformation, especially when integrated near regulatory elements of proto-oncogenes.
Innovation Solution
The development of novel guide nucleic acids (gNAs) and nucleic acid-guided nuclease complexes, such as CRISPR-Cas systems, that target specific genomic sites for precise insertion of exogenous DNA, minimizing disruption to native gene expression and avoiding oncogenic transformation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If random integration methods are used for exogenous gene expression, then integration simplicity is improved, but gene expression safety and stability deteriorate due to disruption of native genes and potential malignant transformation
Solution Approach 1:
The patent applies preliminary action by using guide nucleic acids to pre-identify and target specific safe integration sites in the genome before performing the integration. This preliminary targeting ensures that exogenous genes are inserted only at predetermined safe locations that will not disrupt native gene expression or cause malignant transformation, thereby resolving the contradiction between integration simplicity and gene expression safety
Solution Approach 2:
The patent uses guide nucleic acids as intermediaries that mediate between the exogenous gene and the target genome. These guide nucleic acids direct the integration process to specific safe sites, acting as a mediator that ensures both the simplicity of the integration process and the safety of gene expression by preventing insertion into harmful genomic regions
2Productivity
If random integration is performed near regulatory elements, then integration efficiency is improved, but harmful effects worsen due to disruption of proto-oncogene regulation and malignant transformation
Solution Approach 1:
The patent applies local quality by designating specific local regions in the genome as safe integration sites that have been predetermined to lack harmful regulatory elements. The guide nucleic acids direct integration only to these specific local regions with safe qualities, ensuring high integration efficiency while eliminating the harmful effects of inserting near proto-oncogene regulatory elements
Solution Approach 2:
The patent applies preliminary anti-action by using guide nucleic acids to pre-identify and avoid regions with harmful regulatory elements before integration occurs. This preliminary avoidance action prevents the harmful effects of disrupting proto-oncogene regulation, thereby resolving the contradiction between integration efficiency and malignant transformation risk
Data Source
AI summary
The present invention relates to engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems and corresponding guide RNAs that target specific nucleotide sequences at certain gene loci in the human genome. Also provided are methods of targeting, editing, and/or modifying of the human genes using the engineered CRISPR systems, and compositions and cells comprising the engineered CRISPR systems.