Compact TnpB-Family Nucleases for Viral Vector Genome Editing
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Solution Overview
Problem
Current genome editing technologies, such as CRISPR-Cas9 and Cas12, are large in size, limiting their packaging and application in small viral vectors, and the biological roles of TnpB-family nucleases, evolutionary ancestors to these enzymes, remain enigmatic.
Innovation Solution
Development of engineered systems comprising TnpA, TnpB, and IscB proteins, or their nucleic acids, with optional guide RNAs, derived from Geobacillus stearothermophilus, Clostridium botulinum, and Clostridioides difficile, for targeted DNA modification, including site-specific cleavage and integration, utilizing their smaller size and RNA-guided DNA nuclease capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas9 or Cas12 systems are used for genome editing, then high editing efficiency and precision are achieved, but the large size of these systems limits their packaging in small viral vectors
Solution Approach 1:
The patent extracts the essential nuclease domain from the large CRISPR-Cas9 and Cas12 systems to create smaller TnpB-family nuclease variants that retain genome editing capability while reducing overall system size for viral vector packaging
Solution Approach 2:
The patent creates engineered copies of ancestral TnpB-family nucleases with modified properties, including reduced size and improved stability, that replicate the genome editing function of larger CRISPR systems while being suitable for compact delivery
2Volume of moving object
If TnpB-family nucleases are used for genome editing, then smaller system size enables packaging in viral vectors, but the biological roles of these enzymes remain enigmatic and require engineering
Solution Approach 1:
The patent applies parameter changes to TnpB-family nucleases by modifying amino acid sequences, expression conditions, and structural characteristics to optimize enzymatic activity, stability, and packaging efficiency while maintaining the desired compact size
3Adaptability or versatility
If traditional genome editing systems are used, then comprehensive genome modification capabilities are achieved, but delivery to target cells via viral vectors is limited by system size
Solution Approach 1:
The patent segments the genome editing function into essential and non-essential components, retaining only the core nuclease activity in the compact TnpB system while potentially complementing with separately delivered components for enhanced versatility
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 enable efficient and compact genome editing, suitable for use in small viral vectors, offering diverse applications in genome engineering and potential therapeutic interventions.
Implementation Method 1
engineered systems comprising a TnpA protein, a TnpB protein, an IscB protein, or a combination thereof... for targeted DNA modification, including site-specific cleavage and integration
Implementation Method 2
at least one guide RNA, or one or more nucleic acids encoding thereof, wherein the at least one guide RNA is complementary to at least a portion of a target nucleic acid
Data Source
AI summary
The present disclosure provides systems, compositions, and methods for nucleic acid modification. More particularly, the present disclosure provides systems comprising a TnpA protein, a TnpB protein, an IscB protein, or a combination thereof, and methods using thereof.