Cas9 Cytidine Base Editor Fusion for Precise Single-Nucleotide Editing
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Solution Overview
Problem
Current genome editing technologies suffer from low efficiency and unwanted gene alterations due to stochastic processes like NHEJ and HDR, making precise and efficient single-nucleotide corrections challenging, especially for genetic diseases.
Innovation Solution
A fusion protein comprising a Cas9 domain, a cytidine deaminase domain, and a uracil glycosylase inhibitor (UGI) domain, which can be programmed to efficiently edit specific nucleotides with minimal off-target activity and indel formation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas9 nucleases are used for targeted genome editing, then specific nucleotide changes can be achieved, but low editing efficiency and unwanted gene alterations occur due to stochastic NHEJ and HDR processes
Solution Approach 1:
The patent combines a dead Cas9 (dCas9) domain with a cytidine deaminase domain to create a base editor fusion protein. This merging allows the system to perform direct base conversion (C to U) without relying on stochastic NHEJ or HDR processes, thereby improving both editing precision and efficiency simultaneously. The dCas9 provides targeted binding while the deaminase executes the precise chemical conversion.
Solution Approach 2:
The invention replaces the mechanical DNA cleavage and repair process (NHEJ/HDR) with a direct chemical deamination reaction. Instead of using active Cas9 to create double-strand breaks that require cellular repair machinery, the base editor uses dCas9-bound cytidine deaminase to directly convert C to U through chemical modification, eliminating the stochastic nature of repair processes.
2Reliability
If active Cas9 nucleases are used for genome editing, then targeted cleavage can be achieved, but unwanted indel mutations compete with desired alterations
Solution Approach 1:
The patent extracts the nuclease activity from the Cas9 domain by using dead Cas9 (dCas9) with mutations that abolish cleavage function (e.g., D10A, H840A). This extraction removes the harmful indel-generating cleavage activity while preserving the targeted binding capability, allowing the deaminase domain to perform the desired base conversion without competing indel mutations.
Solution Approach 2:
The invention converts the potentially harmful off-target cleavage activity into a beneficial feature by using dCas9's high-specificity binding without cleavage. The system leverages the precise targeting capability of Cas9 while eliminating its harmful nuclease activity, and combines it with deaminase activity that only modifies the intended base.
3Productivity
If conventional base editing approaches are used, then some nucleotide changes can be achieved, but indel formation remains a significant problem
Solution Approach 1:
The patent merges dCas9 with cytidine deaminase and UGI to create a unified base editor system. This combination allows direct C to U conversion without requiring DNA cleavage or cellular repair pathways, thereby achieving high base editing efficiency while minimizing indel formation. The UGI component further protects against indels by preventing uracil excision that could trigger repair.
Solution Approach 2:
The invention replaces the mechanical DNA cleavage and error-prone repair processes with a clean chemical deamination reaction. The cytidine deaminase directly converts C to U through chemical modification, and the dCas9-U GI complex prevents subsequent excision and repair that would generate indels, achieving high efficiency with minimal harmful byproducts.
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 fusion protein achieves precise and efficient single-nucleotide editing with reduced unintended mutations, providing a powerful tool for gene editing research and therapeutics.
Implementation Method 1
a cytidine deaminase domain... which can be used with a guide RNA to specifically edit nucleic acids by deaminating target cytidine residues
Implementation Method 2
a uracil glycosylase inhibitor (UGI) domain
Implementation Method 3
The clustered regularly interspaced short palindromic repeat (CRISPR) system is a recently discovered prokaryotic adaptive immune system via base-pairing
Data Source
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AI summary
Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing proteins or protein domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing proteins or domains, are provided.