RNA-Guided DNA Integration Without Double-Strand Breaks
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Solution Overview
Problem
Existing CRISPR-Cas systems require DNA double-strand breaks for homology-directed repair, limiting their efficiency and precision in targeted DNA integration.
Innovation Solution
An engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated transposon (CAST) system comprising Cas proteins, transposon-associated proteins, and an unfoldase protein, which facilitates RNA-guided DNA targeting and integration without the need for DNA double-strand breaks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas systems use homology-directed repair requiring DNA double-strand breaks, then targeted DNA integration can be achieved, but efficiency and precision are limited
Solution Approach 1:
The invention extracts and removes the requirement for DNA double-strand breaks from the CRISPR-Cas system by integrating transposon machinery. This extraction of the harmful/limiting element (DSB requirement) allows the system to achieve both high precision through RNA-guided targeting and high efficiency through transposon-mediated integration, resolving the technical contradiction between precision and reliability
Solution Approach 2:
The invention merges CRISPR-Cas RNA-guided targeting machinery with transposon integration machinery into a unified CAST system. This combination allows the system to inherit the precise targeting capability of CRISPR-Cas while gaining the high efficiency of transposon-mediated integration, simultaneously improving both precision and reliability of targeted DNA integration
2Productivity
If CRISPR-Cas systems require DNA double-strand breaks for integration, then genetic payloads can be delivered, but the process becomes more complex and less efficient
Solution Approach 1:
The transposon integration machinery serves multiple functions: it mediates DNA integration, facilitates genetic payload delivery, and eliminates the need for DSB formation. This multi-functionality increases productivity while the modular nature of the CAST system maintains manageable complexity by reusing established biological components
3Measurement precision
If homology-directed repair is used for targeted integration, then genetic modification can occur, but the process is time-consuming and less precise
Solution Approach 1:
The transposon machinery acts as an intermediary that directly mediates DNA integration without requiring the time-consuming homology-directed repair process. The RNA guide serves as an intermediary that provides precise targeting information to the transposon system, enabling both high precision and rapid integration by bypassing the slow HDR pathway
Data Source
AI summary
The present disclosure provides methods and systems for DNA modification and gene targeting comprising an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated transposon (CAST) systems. More particularly, the present disclosure provides systems comprising: an engineered CAST system or one or more nucleic acids encoding the engineered CAST system, wherein the CAST system comprises at least one or both of: a) at least one Cas protein (e.g., Cas6, Cas7, Cas5, and/or Cas8) and b) one or more transposon-associated proteins (e.g., TnsA, TnsB, TnsC, TnsD, and/or TniQ), and at least one unfoldase protein (e.g., ClpX), or a nucleic acid encoding thereof. The present disclosure also provides systems, kits, and methods for nucleic acid modification in a cell.