Chimeric Transposases for Shorter ITR Site-Specific Integration
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Solution Overview
Problem
Current transposases, such as PiggyBac, exhibit asymmetrical binding with their Inverted Terminal Repeat (ITR) sequences, requiring multiple molecules for transposition and longer ITR sequences, while symmetrical dimers like MosI are more efficient but less studied.
Innovation Solution
Development of chimeric transposases combining a truncated Super piggyBac transposase with a C-terminal Cysteine Rich Domain (CRD) deletion and MosI DNA binding domains, optimized with linker sequences and hyperactive mutations, for site-specific transposition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If PiggyBac transposase is used with natural ITR sequences, then transposition can occur, but the ITR sequences are long and multiple transposase molecules are required
Solution Approach 1:
The invention divides the transposase function into two separate components: a catalytic domain (from PiggyBac or related transposases) and a DNA binding domain (from Mos1 or other transposases). This segmentation allows the DNA binding domain to recognize and bind to shortened ITR sequences, while the catalytic domain performs the transposition reaction, thereby reducing the required ITR length while maintaining transposition efficiency.
Solution Approach 2:
The invention merges the catalytic activity of PiggyBac transposase with the DNA binding capability of Mos1 transposase into a chimeric transposase protein. This fusion protein combines the strengths of both parent transposases: the efficient catalysis of PiggyBac and the symmetrical dimer binding of Mos1, enabling effective transposition with shorter ITR sequences and reduced transposase molecule requirements.
2Productivity
If PiggyBac transposase binds asymmetricalIy to ITR sequences, then transposition can occur, but multiple transposase molecules are needed
Solution Approach 1:
The invention adopts the symmetrical dimer binding mode of Mos1 transposase, which binds to ITR sequences in a symmetrical manner. This symmetrical binding allows two transposase molecules to efficiently recognize and bind to the ITRs simultaneously, reducing the overall quantity of transposase molecules needed compared to the asymmetrical binding mode of native PiggyBac transposase.
3Reliability
If multiple transposase molecules are used for transposition, then the reaction can proceed, but the complexity of the system increases
Solution Approach 1:
The chimeric transposase is designed to perform multiple functions within a single protein: DNA binding, dimerization, and catalytic activity. The N-terminal domain handles DNA recognition and binding to the ITR sequences, while the C-terminal domain provides the catalytic function for transposition. This multi-functionality reduces system complexity by eliminating the need for separate binding and catalytic components.
Data Source
AI summary
Provided herein are chimeric transposases and chimeric site-specific fusion proteins, polynucleotides encoding the chimeric transposases and chimeric site-specific fusion proteins, and vectors and transposons comprising the polynucleotides. Also provided are methods of making the chimeric transposases and chimeric fusion proteins, cells that are modified using the chimeric transposases or chimeric site-specific fusion proteins provided herein and methods using such cells.