Sleeping Beauty Transposase Variants for Higher DNA Insertion
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current transposon systems, particularly the Sleeping Beauty (SB) system, face limitations in transpositional activity and insertion efficiency, which restrict their effectiveness in gene transfer and mutagenesis applications.
Innovation Solution
Development of hyperactive variants of the SB10 transposase with specific amino acid mutations, such as K14R, K33A, R214D, and others, enhancing transposase activity by up to 100-fold compared to the natural SB10.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the natural SB10 transposase is used, then the system is simple and easy to implement, but the transpositional activity and insertion efficiency are limited
Solution Approach 1:
The patent applies parameter changes by systematically mutating specific amino acid residues in the SB10 transposase protein sequence. Multiple point mutations were introduced at positions including 14, 33, 115, 143, 147, 205, 207, 208, 210, 214, 215, 216, 217, 243, 267, 314, and 317 to optimize the protein's catalytic activity and DNA binding properties, thereby enhancing transpositional activity while maintaining the basic protein structure.
Solution Approach 2:
The patent creates composite functional elements by combining multiple hyperactive mutations within the transposase protein to generate variants with synergistic effects. The hyperactive transposase variants integrate multiple beneficial amino acid substitutions that work together to achieve up to 100-fold enhancement in transpositional activity compared to the wild-type SB10 transposase.
2Productivity
If the natural SB10 transposase is used, then the system is easy to prepare, but the insertion efficiency into target DNA is limited
Solution Approach 1:
The patent optimizes the transposase protein's functional parameters by introducing specific amino acid mutations that enhance DNA binding affinity and catalytic efficiency. The mutated variants exhibit improved ability to recognize and process target DNA sequences, resulting in significantly enhanced insertion efficiency while the protein can still be produced using standard molecular biology techniques.
3Adaptability or versatility
If more target loci need to be mutagenized, then the coverage and applicability increase, but the current transposase activity is insufficient
Solution Approach 1:
The patent enhances the transposase's catalytic parameters through rational mutagenesis, creating variants with increased specific activity that can process multiple target loci simultaneously. The hyperactive variants enable comprehensive genome-wide mutagenesis and transposition events across numerous target sites, thereby expanding the system's versatility for functional genomics applications.
Solution Approach 2:
The patent performs preliminary optimization of the transposase protein in vitro by introducing hyperactive mutations before application in vivo. This pre-optimization ensures that the enhanced transposase variants are ready to efficiently mutagenize multiple target loci when introduced into cells, maximizing genome coverage without requiring excessive amounts of transposase protein.
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 mutated SB10 transposase variants significantly improve DNA insertion efficiency and transpositional activity, allowing for more effective gene transfer and mutagenesis by increasing the number of target loci that can be mutagenized.
Implementation Method 1
Active transposons encode enzymes that facilitate the excision and insertion of the nucleic acid into target DNA sequences. Class II transposons move by a cut and paste mechanism, rather than by copy and paste, and use the transposase enzyme in this mechanism.
Data Source
AI summary
The present invention refers to hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB). The invention further refers to corresponding nucleic acids producing these variants, to a gene transfer system for stably introducing nucleic acid(s) into the DNA of a cell by using these hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB) and to transposons used in the inventive gene transfer system, comprising a nucleic acid sequence with flanking repeats (IRs and/or RSDs). Furthermore, applications of these transposase variants, the transposon, or the gene transfer system are also disclosed such as gene therapy, insertional mutagenesis, gene discovery (including genome mapping), mobilization of genes, library screening, or functional analysis of genomes in vivo and in vitro. Finally, pharmaceutical compositions and kits are also encompassed.