Cas12a Circular Permutant for Expanded Nucleic Acid Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Cas12a and base editors with a Cas12a domain have a limited editing window, necessitating the development of new enzymes with enhanced nucleic acid editing capabilities.
Innovation Solution
A polypeptide comprising a circular permutant of Cas12a is developed, which binds to nucleic acids and possesses nuclease activity. This circular permutant can be fused with polypeptides of interest, such as deaminases and reverse transcriptases, to enhance editing capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If Cas12a and base editors with a Cas12a domain are used, then nucleic acid binding and nuclease activity are achieved, but the editing window is limited
Solution Approach 1:
The patent applies segmentation by creating circular permutants that divide the protein structure into distinct functional domains. The circular permutant of Cas12a is constructed by cutting the protein at specific locations and rejoining at different positions, creating multiple functional segments that can independently bind to nucleic acids and provide nuclease activity. This segmentation allows the enzyme to achieve a wider editing window while maintaining reliable editing efficiency through the coordinated action of these segmented domains.
2Productivity
If circular permutant of Cas12a is used, then editing efficiency is improved, but enzyme structure complexity increases
Solution Approach 1:
The patent applies dynamics by creating a circular permutant structure that can dynamically adjust its conformation during the editing process. The circular permutant of Cas12a features a flexible protein structure that can transition between different states to facilitate nucleic acid binding, catalysis, and product release. This dynamic structure allows the enzyme to achieve high editing efficiency while managing structural complexity through controlled conformational changes rather than rigid fixed structures.
Solution Approach 2:
The patent applies parameter changes by modifying the protein sequence parameters through circular permutation. The circular permutant is generated by changing the amino acid sequence parameters - specifically, by cutting the protein at different positions and rejoining at different locations, which alters the protein's structural and functional parameters. This parameter change approach enables improved editing efficiency while managing structural complexity through systematic sequence modification rather than random structural changes.
3Measurement precision
If circular permutant binds to nucleic acid, then editing specificity is enhanced, but off-target effects may increase
Solution Approach 1:
The patent applies local quality by creating circular permutants with region-specific functional properties. The circular permutant of Cas12a is constructed with different domains that have specialized local qualities - some regions are optimized for high-specificity nucleic acid binding, while other regions provide nuclease activity with controlled specificity. This local quality differentiation allows the enzyme to enhance editing specificity at target sites while minimizing off-target effects through the specialized properties of individual protein domains.
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 circular permutant of Cas12a demonstrates improved nucleic acid editing efficiency, as evidenced by increased indel percentages and precise editing percentages at target nucleic acid sites compared to control enzymes.
Implementation Method 1
the circular permutant binds to a nucleic acid
Implementation Method 2
the circular permutant has nuclease activity
Data Source
AI summary
Described herein are circular permutants along with compositions and systems comprising the circular permutants. Fusion proteins including a circular permutant are also described such as fusion proteins that include a circular permutant fused to a polypeptide of interest (e.g., a deaminase or reverse transcriptase). Also described herein are methods of using and/or producing a circular permutant such as methods of using a circular permutant for modifying or editing a target nucleic acid.