Trackable Nucleic Acid Editing via CREATE Cassette
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Solution Overview
Problem
Current methods for tracking nucleic acid-guided editing 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 nickase/reverse transcriptase fusion enzyme and a CRISPR-enabled trackable genome engineering (CREATE) fusion editing cassette, which integrates a repair template and guide RNA into the cell genome for efficient tracking of genomic edits.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current tracking methods are used to track nucleic acid-guided editing, then tracking capability is provided, but integration efficiency is low and random genomic integration occurs
Solution Approach 1:
The patent introduces a tracking cassette as an intermediary element that mediates between the editing process and tracking requirement. This cassette contains a guide RNA sequence that directs the nuclease to a specific integration site, serving as a bridge that enables both efficient tracking and precise genomic integration without random insertion
Solution Approach 2:
The tracking cassette is designed and prepared in advance with predetermined integration sites and guide sequences. By performing the tracking setup beforehand, the system eliminates the need for successive rounds of editing and ensures direct, efficient integration in a single editing event
2Manufacturing precision
If successive rounds of editing are performed for targeted integration, then integration precision is improved, but time consumption increases
Solution Approach 1:
The tracking cassette is pre-designed with specific integration site sequences and corresponding guide RNA elements before the editing process begins. This preliminary preparation allows the system to achieve precise targeted integration in a single editing round, eliminating the need for multiple successive editing cycles and significantly reducing time consumption
3Reliability
If barcode sequences are added for tracking, then tracking capability is enhanced, but system complexity increases
Solution Approach 1:
The tracking cassette is designed as a multi-functional element that combines several functions into a single component: it contains the guide RNA sequence for directing nuclease activity, the tracking sequence for monitoring integration, and the structural elements for stable genomic incorporation. This universal design eliminates the need for separate barcode sequences and associated delivery systems, reducing overall system complexity while maintaining enhanced tracking capability
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 tracking of genomic edits in live cells by integrating a trackable cassette into the genome, simplifying the editing process and eliminating the need for additional barcode sequences.
Implementation Method 1
nucleic acid-guided nickase/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.