Single Nucleic Acid Construct for Large Site-Specific Gene Integration
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Solution Overview
Problem
Current genome editing techniques require double strand breaks, which evoke DNA damage responses and are inactive in terminally differentiated cells, limiting the insertion and deletion of large sequences, and CRISPR-based approaches are limited to short sequence modifications.
Innovation Solution
A single nucleic acid construct incorporating a prime editor system, guide RNAs, integrases, and recombinases, capable of self-circularizing and integrating large DNA sequences into any genomic locus using various delivery vectors, enabling efficient and multiplexed genome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If double strand breaks are used for genome integration, then DNA repair pathways are activated, but the process becomes inactive in terminally differentiated cells and evokes DNA damage responses
Solution Approach 1:
The patent introduces integrase enzymes as intermediary proteins that mediate DNA integration without requiring double-strand breaks. The integrase recognizes specific attB and attP sites and catalyzes recombination directly, bypassing the need for DNA damage response pathways that are inactive in terminally differentiated cells.
Solution Approach 2:
The patent replaces the mechanical breakage-repair mechanism (double-strand breaks followed by repair pathways) with a chemical/enzymatic recombination mechanism (integrase-mediated site-specific recombination). This substitution allows genome integration to proceed in cell types where DNA repair pathways are inactive.
2Reliability
If CRISPR-based approaches are used to bypass double stranded breaks, then short sequence modifications are achieved, but insertion and deletion of large sequences is limited
Solution Approach 1:
The patent segments the DNA cargo into manageable pieces that can be packaged with integrase and guide RNAs. The system uses multiple guide RNAs (gRNA1, gRNA2, gRNA3) that can target different locations, allowing stepwise integration of larger DNA sequences through multiple recombination events rather than requiring single-step insertion of entire large sequences.
Solution Approach 2:
The patent adds the dimension of time by enabling multi-step integration processes. Instead of attempting to insert large sequences in a single event, the system performs sequential integrations at different time points, with each step integrating a portion of the total cargo. This temporal dimension allows accumulation of large DNA sequences through multiple smaller events.
3Reliability
If multiple gene editing components are delivered separately, then each component can be optimized, but the delivery process becomes complex and less efficient
Solution Approach 1:
The patent merges multiple gene editing components (integrase enzyme, guide RNAs, and DNA cargo) into a single integrated delivery construct. This single construct contains all necessary elements for site-specific recombination and can be delivered as one unit, simplifying the delivery process while maintaining the functionality of each individual component.
Solution Approach 2:
The patent creates a universal delivery system that can handle various DNA cargos and target different genomic locations using the same integrase-cargo construct architecture. The system is multi-functional, capable of integrating different sequences at different loci without requiring separate optimized delivery systems for each application.
Data Source
AI summary
The present disclosure provides nucleic acid compositions, methods, and an overall platform for site-specific genetic engineering using Programmable Addition via Site-Specific Targeting Elements (PASTE), transposon-mediated gene editing, or other suitable gene editing or gene incorporation technology packaged into a single nucleic acid construct.