Modified PBase–Cas9 Fusion for Targeted Long-Gene Integration
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Solution Overview
Problem
Current gene therapy methods face challenges in efficiently integrating exogenous DNA sequences, particularly long fragments, into specific genomic loci with high integration efficiency and low off-target rates, using existing PBase mutants and CRISPR Cas systems.
Innovation Solution
A modified PBase protein with specific amino acid mutations, combined with site-specific DNA nucleases, enhances the ability to integrate long foreign genes at double-strand breaks, reducing off-target integration and simplifying donor DNA preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR Cas system mediated homologous recombination is used to achieve targeted integration, then integration specificity is improved, but integration efficiency and fragment length are worsened
Solution Approach 1:
The patent merges the targeted DNA cleavage capability of CRISPR-Cas9 with the transposition integration capability of PBase enzyme. The fusion protein combines both functional domains, allowing the system to first create targeted double-strand breaks at specific genomic loci via Cas9, then efficiently integrate transposon-carried exogenous genes through PBase-mediated transposition, thereby achieving both high specificity and high efficiency simultaneously
Solution Approach 2:
The invention creates a composite enzymatic system by fusing Cas9 nuclease and PBase transposase into a single fusion protein. This composite structure integrates the guide RNA-directed targeting mechanism of CRISPR with the transposon integration mechanism of PiggyBac, enabling the system to leverage both mechanisms' strengths for targeted gene integration
2Productivity
If PBase enzyme is used to integrate transposon DNA into TTAA site, then integration efficiency is improved, but off-target integration rate is worsened
Solution Approach 1:
The fusion protein acts as an intermediary that couples the specificity of CRISPR-Cas9 targeting with the efficiency of PBase transposition. The Cas9 domain ensures that DNA cleavage occurs only at the intended target locus guided by sgRNA, while the PBase domain mediates the actual transposon integration, thereby preventing off-target integration events that would occur with PBase alone
3Manufacturing precision
If linear donor DNA with homologous arms is prepared for homologous recombination, then targeted integration is improved, but preparation time and cost are worsened
Solution Approach 1:
The invention extracts the requirement for complex linear donor DNA with homologous arms by utilizing circular transposon donor plasmids instead. The transposon structure with inverted terminal repeats (ITR) replaces the need for long homologous arms, dramatically simplifying donor DNA preparation while maintaining targeted integration capability through the fusion protein system
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
The modified PBase protein achieves efficient targeted insertion of long genes up to 12kb, with reduced off-target integration and lower costs, using circular plasmids as donors, improving gene therapy efficacy.
Implementation Method 1
The PBase protein retains the ability to bind to the ITR sequence of the Piggybac transposon and cleaves it to isolate the Piggybac transposon from the donor plasmid in a linear manner
Implementation Method 2
combining the corresponding 'Exc+Int-' mutant with Cas9 gRNA can integrate Transposon DNA at specific sites guided by gRNA
Implementation Method 3
CRISPR Cas mediated DSB can achieve targeted integration of exogenous genes through homologous recombination (HR) pathway repair
Data Source
Figure 1A~1B
Figure 2~3A
Figure 3B
AI summary
Provided in the present invention are a PBase protein, a fusion protein, a nucleic acid, a gene integration system, a gene delivery system and the use. The PBase protein retains the abilities of binding to the ITR sequences of a Piggybac transposon and cleaving the Piggybac transposon to make the Piggybac transposon separate from a donor plasmid in a linear manner; and the PBase protein has a lower ability for binding to the double strands of DNA within 10 bp upstream and downstream of "TTAA". When the PBase protein is linked to a site-specific DNA nuclease to prepare the fusion protein, the PBase protein can be efficiently integrated with a long fragment gene of interest at the double strand breaks generated by the site-specific DNA nuclease, thereby achieving the effect of site-directed insertion of the long fragment gene.