Split Complementary Base Editing Systems Reduce Off-Target Mutations
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Solution Overview
Problem
Traditional base editing systems, such as BE3, often induce significant random off-target mutations at a genome-wide level, posing safety risks and uncertainties, especially in clinical applications.
Innovation Solution
The development of split complementary base editing systems, which utilize complementary bimolecular deaminases, significantly reduces off-target effects while maintaining robust on-target editing activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional base editing systems (such as BE3) are used, then on-target editing activity is achieved, but random off-target mutations are induced at genome-wide level
Solution Approach 1:
The base editor is divided into two separate proteins: a Cas9 nickase component that binds to the target site via gRNA, and a deaminase component that performs the base conversion. This segmentation ensures that the deaminase is only activated when the Cas9-nickase is properly bound to the target sequence, thereby eliminating random off-target deamination events while maintaining on-target editing activity.
Solution Approach 2:
The Cas9-nickase acts as an intermediary that mediates between the gRNA and the deaminase. It forms a ribonucleoprotein complex with gRNA that specifically recognizes the target sequence, and only then recruits or activates the deaminase component. This intermediary mechanism ensures spatial and temporal control of the deamination reaction, preventing off-target effects.
2Reliability
If base editing systems are used for clinical applications, then genetic diseases can be treated, but safety risks arise from unpredictable off-target effects
Solution Approach 1:
By segmenting the base editor into separate Cas9-nickase and deaminase components, the system ensures that the potentially harmful deaminase activity is strictly controlled and only occurs when the Cas9-nickase is properly bound to the intended target site. This physical separation eliminates the possibility of the deaminase acting on off-target sites independently.
Solution Approach 2:
The system changes the operational parameters of the base editing machinery by requiring dual recognition: the Cas9-nickase must recognize the target sequence through gRNA binding, and only then can the deaminase component function. This parameter change from a single-component to a two-component system fundamentally alters the specificity and safety profile of the editing process.
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
These systems achieve near-background levels of genome-wide off-target edits, enhancing the safety and efficacy of genome editing, particularly in crop genetic improvement and clinical treatment of human genetic diseases.
Implementation Method 1
the fused cytidine deaminase rAPOBEC1 catalyzes cytosine (C) bases into uracil (U) bases at a specific site in a target sequence
Data Source
AI summary
Disclosed are split complementary base editing systems based on bimolecular deaminases and uses thereof. A split complementary base editing system mainly includes base editing fusion proteins A and B that are from splitting at a deaminase domain embedded inside a Cas9 nickase (nCas9), and a guide RNA (gRNA). The present disclosures of split complementary cytosine base editing systems can greatly reduce Cas9-dependent and Cas9-independent off-target effects in the genome while maintaining robust on-target cytosine base editing. The present disclosures also provide the split complementary adenine base editing systems. The split complementary cytosine (adenine) base editing systems are widely applicable to various eukaryotic organisms, and can be used in crop genetic breeding, animal breed improvement, and even clinical treatment of human genetic diseases.