ecDNA Labeling With dCas9 Breakpoint Junction Targeting
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Solution Overview
Problem
Current methods are inadequate for detecting and tracking extrachromosomal DNA (ecDNA) in cancer cells, which are associated with cancer growth and treatment resistance, and there is a need for tools to visualize and target these DNA fragments for diagnostic and therapeutic purposes.
Innovation Solution
The use of a programmable nuclease-based system, such as RNA-guided nucleases like Cas9 or catalytically-inactive Cas9 (dCas9), combined with guide RNA (gRNA) and Pumilio-FBF (PUF) domain binding, to target and label ecDNA with detectable molecules like fluorescent proteins, enabling visualization and molecular cytogenetic tracing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods are used, then general DNA detection is possible, but ecDNA-specific detection and visualization cannot be achieved
Solution Approach 1:
The patent applies local quality by designing gRNAs that specifically target unique breakpoint junction sequences at specific locations within ecDNA, rather than targeting general DNA sequences. This localized targeting approach enables precise differentiation of ecDNA from chromosomal DNA, achieving both detection specificity and visualization capability through the expression of detectable molecules at the targeted ecDNA locations
Solution Approach 2:
The patent uses an intermediary approach by introducing RNA-guided nucleases (such as dCas9 fused to detectable molecules) that act as mediators between the target ecDNA sequences and the detection system. These intermediaries bind to specific ecDNA breakpoint junctions and bring detectable molecules to the target sites, enabling visualization without directly modifying the ecDNA structure
2Adaptability or versatility
If catalytically active nucleases are used to target ecDNA, then ecDNA can be cleaved and modified, but ecDNA integrity is compromised
Solution Approach 1:
The patent applies inversion by using catalytically inactive (dead) Cas9 (dCas9) instead of active Cas9. By inverting the nuclease activity while retaining the DNA-binding capability guided by gRNA, the system achieves versatile ecDNA targeting through breakpoint junction recognition while preserving ecDNA structural integrity by preventing cleavage. The detectable molecules are delivered through this inactive complex rather than through cleavage and repair mechanisms
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
Enables precise detection and tracking of ecDNA in cancer cells, providing insights into cancer evolution, treatment resistance, and recurrence, and allowing for diagnostic and therapeutic interventions.
Implementation Method 1
a guide RNA (gRNA) linked to a detectable molecule... wherein the gRNA comprises (a) a targeting sequence that is complementary to the breakpoint junction
Implementation Method 2
a catalytically-inactive RNA-guided nuclease complexed with a guide RNA (gRNA)
Implementation Method 3
a PUF domain linked to the detectable molecule, and the PUF domain binds to the PBS
Implementation Method 4
linked to a detectable molecule... enabling the targeting and/or detection (e.g., visualization) of extrachromosomal DNA (ecDNA)
Data Source
AI summary
Provided herein are methods and tools for targeting detecting (e.g., imaging) extrachromosomal DNA, for example, in cancer cells.