Split Cas12b Polypeptides for Compact Genome Editing
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Solution Overview
Problem
Current CRISPR-Cas systems face limitations in efficiency, ease of use, stability, and specificity for genome editing, necessitating improved methods and systems for effective genome editing.
Innovation Solution
Engineered CRISPR-Cas systems comprising split Cas12b polypeptides, where a reference Cas12b protein is divided into N-terminal and C-terminal portions, and a guide RNA, allowing association to form a complex that specifically binds to a target nucleic acid, with options for dimerization domains and functional domains for enhanced functionality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a full-length Cas12b protein is used for genome editing, then the system provides complete nuclease activity and target binding, but the construct size is large making delivery difficult
Solution Approach 1:
The patent divides the full-length Cas12b protein into two separate polypeptides: a first polypeptide comprising an N-terminal portion and a second polypeptide comprising a C-terminal portion. These separated polypeptides can be delivered independently through smaller vectors while still reconstructing the functional nuclease activity when brought together in the cell, thus resolving the contradiction between maintaining complete functionality and reducing construct size for delivery.
2Productivity
If a full-length Cas12b protein is used, then the system achieves high genome editing efficiency, but the system lacks flexibility for transcriptional regulation
Solution Approach 1:
By segmenting the Cas12b protein into separate N-terminal and C-terminal portions, the patent enables independent modification of each polypeptide with different functional domains. The first polypeptide can be engineered for transcriptional activation while the second maintains nuclease activity, or vice versa, allowing the same system framework to serve multiple functions including both genome editing and transcriptional regulation without requiring separate protein constructs.
Solution Approach 2:
The split polypeptide system provides a universal platform where different functional domains can be independently attached to either the first or second polypeptide. This allows a single CRISPR-Cas system to perform multiple functions such as genome editing, transcriptional activation, and transcriptional repression by simply changing which functional domains are attached to which polypeptide, thereby achieving both high editing efficiency and regulatory flexibility.
3Stability of the object's composition
If a full-length Cas12b protein is used, then the system maintains structural integrity, but the system is difficult to control for multiplex editing
Solution Approach 1:
The segmentation of Cas12b into separate polypeptides allows independent control of each component's expression and localization. Each polypeptide can be separately tagged with localization signals or controlled by independent promoters, enabling precise temporal and spatial control over when and where the reconstituted nuclease becomes active. This controlled reconstitution is particularly useful for multiplex editing where different targets need to be edited at different times or in different cellular compartments.
Data Source
AI summary
Provided are engineered split Cas12b systems and methods of use thereof. Also provided are compositions comprising one or more components of the engineered split Cas12b systems, as well as engineered cells and non-human animals produced by the methods. The systems, methods and compositions are useful for genome editing, transcription modulation and gene therapy.