SECURE-ABE Variants Reduce Off-Target RNA Editing
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Solution Overview
Problem
Adenine base editors (ABEs) face challenges with unwanted off-target RNA editing activity, which can lead to unintended genetic modifications and heterogeneity in base editor coding sequences, complicating their use in research and therapeutic applications.
Innovation Solution
Development of SECURE-ABE variants with specific amino acid substitutions in the E. coli TadA domain to reduce RNA editing activity while preserving DNA editing capability, utilizing mutations at defined positions to minimize off-target RNA editing while maintaining on-target DNA editing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild type E. coli TadA or engineered TadA variants are used in adenine base editors, then DNA editing activity is achieved, but off-target RNA editing activity occurs causing unwanted genetic modifications
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions at defined positions in the TadA domain (such as positions 10, 11, 13, 17, 20, 21, 23, 25, 26, 48, 56, 58, 71, 72, 74, 77, 82, 106, 107, 108, 109, 110, 111, 122, 123, 128, 129, 138, 139, 140, 142, 143, 148, 150, 153, 155). These substitutions modify the biochemical parameters of the adenosine deaminase to reduce its activity toward RNA substrates while preserving its activity toward DNA substrates, thereby resolving the contradiction between DNA editing effectiveness and RNA editing specificity
Solution Approach 2:
The patent applies local quality by making targeted amino acid substitutions at specific positions within the TadA domain rather than uniformly modifying the entire protein. Each substitution is strategically placed to affect local interactions with RNA substrates while leaving DNA binding and catalysis largely intact. This localized modification approach allows the protein to exhibit different functional qualities toward RNA versus DNA substrates
2Object-generated harmful factors
If adenosine deaminase variants with reduced RNA editing activity are introduced, then off-target RNA editing is minimized, but DNA editing efficiency may be compromised
Solution Approach 1:
The patent carefully selects amino acid substitutions that specifically alter the enzyme's substrate preference parameters without significantly impacting its catalytic efficiency toward DNA. The substitutions are chosen to reduce RNA binding affinity or catalytic rate while maintaining DNA processing capability, thus resolving the contradiction between reducing harmful RNA editing and preserving beneficial DNA editing productivity
Solution Approach 2:
The patent creates variant copies of the TadA domain with specific amino acid substitutions that replicate the desired function of reduced RNA editing activity while maintaining DNA editing capability. These engineered copies are then fused to the Cas9 nickase to create functional base editors with improved specificity profiles
3Reliability
If multiple amino acid substitutions are introduced to reduce RNA editing, then specificity is improved, but protein complexity and manufacturing difficulty increase
Solution Approach 1:
The patent identifies a finite set of amino acid positions that, when substituted, produce the desired reduction in RNA editing activity. By focusing modifications on these specific positions rather than conducting broad random mutagenesis, the patent simplifies the manufacturing process while achieving the reliability improvement. The defined substitution positions make the engineering process more predictable and manufacturable
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 SECURE-ABE variants exhibit significantly reduced unwanted RNA editing while retaining robust on-target DNA editing, addressing the issue of genetic heterogeneity and improving the precision of base editors for research and therapeutic applications.
Implementation Method 1
engineered adenosine deaminases that can induce programmable adenosine (A) to inosine (I) edits in single-stranded DNA
Data Source
AI summary
Engineered adenine base editor (ABE) variants with reduced RNA editing activity, and methods of using the same.