Genomic Barcoding via Split Locus Integration
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Solution Overview
Problem
Current methods for tracking nucleic acid-guided editing events in live cells, particularly mammalian cells, are inefficient and may lead to random genomic integration of tracking sequences or require successive rounds of editing for targeted integration.
Innovation Solution
A method involving a nucleic acid-guided nuclease/reverse transcriptase fusion enzyme, combined with CF editing and barcoding cassettes, allows for precise editing and tracking of genomic edits by integrating a barcode into a separate target locus, enabling efficient tracking through genomic sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current tracking methods are used, then tracking of editing events is possible, but the methods are inefficient and lead to random genomic integration
Solution Approach 1:
The method separates the editing function from the tracking function by using distinct gRNAs and repair templates. The editing gRNA targets the first locus for editing while the tracking gRNA targets a second locus for barcode integration, allowing independent optimization of each function and eliminating random integration issues.
Solution Approach 2:
The patent introduces a barcode sequence as an intermediary marker that is integrated at a separate target locus from the editing site. This intermediary tracking marker can be detected through sequencing to identify cells with successful editing, providing reliable tracking without affecting the editing process itself.
2Manufacturing precision
If successive rounds of editing are performed for targeted integration, then integration precision is improved, but the complexity and time required increase
Solution Approach 1:
The method performs preliminary targeting and integration in a single step by providing both the editing gRNA and tracking gRNA along with their respective repair templates simultaneously. This preliminary preparation allows targeted integration to occur in one editing round rather than requiring multiple successive rounds.
3Loss of information
If tracking sequences are integrated into the genome, then tracking is enabled, but random integration reduces tracking reliability
Solution Approach 1:
The patent applies local quality by providing locus-specific gRNAs and repair templates for both editing and tracking. The editing gRNA is designed to target a specific first locus while the tracking gRNA targets a different second locus, ensuring that tracking occurs at a predetermined location rather than randomly throughout the genome.
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
This approach enables efficient and precise tracking of genomic edits in live cells, reducing the complexity and inefficiency of current methods, and allowing for the analysis of editing events through barcode sequencing.
Implementation Method 1
nucleic acid-guided nuclease/reverse transcriptase fusion enzyme
Data Source
AI summary
The present disclosure provides compositions of matter, methods and instruments for nucleic acid-guided nickase/reverse transcriptase fusion enzyme editing of nucleic acids in live mammalian cells, and for tracking of editing events.