RNA-Mediated Base Editing System for Precise Genome Modification
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Solution Overview
Problem
Current gene editing methods, particularly CRISPR-Cas systems, face challenges in precision and safety, especially in multiplexed gene editing, with high variability in repair outcomes and potential genotoxicity, necessitating a more controlled and efficient approach for genetic modification in human cells.
Innovation Solution
A novel RNA-mediated base editing system that allows for the reversible recruitment of multiple effectors to a single genomic locus, enabling efficient and precise base editing without inducing double-stranded breaks, using a guide RNA and ligand binding complex to associate with effector proteins, allowing for simultaneous or sequential editing at multiple sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR-Cas systems induce double-stranded breaks for gene editing, then genome alterations can be achieved, but genotoxicity and chromosomal translocations occur
Solution Approach 1:
The patent extracts and removes the DNA cleavage function from the CRISPR-Cas system by using nuclease-deficient Cas9 variants (dCas9), thereby eliminating the harmful double-stranded breaks while retaining the targeted DNA binding capability for precise base editing
Solution Approach 2:
The patent introduces base editing enzymes (such as cytidine deaminase and adenine deaminase) as intermediary molecules that enable precise nucleotide conversion without requiring DNA cleavage, thus achieving genome editing without genotoxicity
2Adaptability or versatility
If multiple double-stranded breaks are induced for multiplexed gene editing, then multiple genes can be edited, but chromosomal translocations and oncogenic effects increase
Solution Approach 1:
The patent removes the DNA cleavage function entirely from multiplexed editing operations by using dCas9-based base editing systems, allowing multiple target sites to be edited simultaneously without inducing chromosomal breaks or translocations
Solution Approach 2:
The patent replaces the mechanical DNA breakage and repair mechanism with a chemical base conversion mechanism mediated by deaminase enzymes, enabling multiplexed editing without the harmful effects of multiple DSBs
3Manufacturing precision
If homology directed repair is used for precise nucleotide alteration, then accurate editing can be achieved, but editing efficiency is low
Solution Approach 1:
The patent extracts and eliminates the requirement for homology directed repair by using base editing enzymes that directly convert nucleotides through chemical modification, bypassing the inefficient HDR pathway while maintaining high precision
Solution Approach 2:
The patent replaces the complex HDR repair mechanism with a simplified enzymatic base conversion process, dramatically improving editing efficiency while preserving nucleotide-level precision
4Ease of manufacture
If non-homologous end joining is used for gene disruption, then simple gene knockout can be achieved, but repair outcome variability is high
Solution Approach 1:
The patent removes the reliance on non-homologous end joining by using base editing to directly convert nucleotides, eliminating the variability inherent in NHEJ repair outcomes while maintaining operational simplicity
Solution Approach 2:
The patent replaces the stochastic NHEJ repair process with a deterministic enzymatic base conversion reaction, ensuring consistent and predictable editing outcomes
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 system enhances editing efficiency and specificity, reduces off-target effects, and allows for the use of various deaminases to perform both cytidine and adenosine edits simultaneously, improving the precision and safety of genetic modifications.
Implementation Method 1
Directed by a guide RNA (gRNA), CRISPR-Cas systems induce a double stranded break (DSB) within a gene of interest
Implementation Method 2
recruits a base editing enzyme to a target DNA sequence through the guide RNA component of a CRISPR-Cas complex
Data Source
Figure 1
Figure 2(A)~2(H)
Figure 3(A)~3(H)
AI summary
The present invention relates to an RNA-mediated base editing system that enables recruitment of multiple effector proteins to a single target site, allowing for highly efficient base editing. The invention further relates to methods and kits for genetically modifying a cell.