Cas12a Adenine Base Editors With Broader Target Range and Specificity
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Solution Overview
Problem
Current adenine base editors (ABEs) based on Cas9 have limitations in target range and specificity, and Cas12a-based ABEs with high activity and specificity are not yet available for applications in plants, leading to challenges in precision genome editing.
Innovation Solution
Development of Cas12a-based ABEs with a monomeric structure and a simplified domain architecture, optimized through iterative testing of nuclear localization signal, linker, and adenosine deaminase domain components, to enhance versatility and efficiency in various host cells, particularly in plants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If Cas9-based ABEs are used, then adenine base editing function is achieved, but target range and specificity are limited
Solution Approach 1:
The patent changes the nuclease platform from Cas9 to Cas12a, fundamentally altering the PAM recognition parameters. Cas12a recognizes T-rich PAM sequences (5'-TTTV-3') instead of Cas9's NGG PAM, thereby expanding the target range to regions previously inaccessible to Cas9-based editors while maintaining editing specificity through the distinct PAM dependency of Cas12a
2Adaptability or versatility
If Cas12a-based ABEs are developed, then broader target range is achieved, but activity and specificity remain insufficient
Solution Approach 1:
The patent applies local quality optimization by making site-specific improvements to the Cas12a protein structure. Multiple point mutations are introduced at specific positions within the Cas12a domain to enhance its catalytic activity and binding specificity, thereby improving overall editor performance without altering the fundamental Cas12a architecture or its PAM recognition properties
Solution Approach 2:
The patent creates a composite editor by fusing the optimized Cas12a nuclease domain with the TadA8e adenosine deaminase domain. This composite structure combines the target recognition and binding capabilities of Cas12a with the adenine deamination function of TadA8e, achieving both broad target range through Cas12a's PAM flexibility and high editing specificity through the coordinated action of both domains
3Ease of operation
If domain architecture is simplified to enhance versatility, then ease of operation is improved, but structural complexity reduction may affect function
Solution Approach 1:
The patent extracts and removes unnecessary domains from the editor architecture. By eliminating redundant or non-essential domains while retaining the core Cas12a nuclease domain and TadA8e deaminase domain, the patent simplifies the overall structure, facilitating easier delivery and operation in diverse host cells while preserving the essential base editing function through the streamlined domain composition
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 optimized Cas12a-based ABEs demonstrate high, targeted, and specific activity with equal or better editing efficiency compared to existing ABEs, suitable for diverse applications in plants and other organisms.
Implementation Method 1
catalyze adenine into inosine, which is repaired as guanine, leading to A-T to G-C transitions
Data Source
AI summary
The present invention relates to an adenine base editor (ABE), and components thereof. The present invention also relates to a complex comprising an adenine base editor (ABE) and a guide RNA in a functionally associated form. The present invention further relates to a nucleic acid molecule encoding the ABE/guide RNA, an expression construct or a vector comprising a nucleic acid sequence encoding the adenine base editor and/or the nucleic acid sequence encoding the guide RNA. The present invention further relates to a cell comprising an adenine base editor (ABE) and a method of adenine base editing of a target site in a genome of interest in at least one cell of a prokaryotic organism, including bacterial and archaeal organisms, or eukaryotic organism. Besides that, the present invention relates to various methods, kits and uses associated with the ABEs provided.