Engineered Sleeping Beauty transposase redirects transposon integration away from genes
Csaba Miskey1, Lisa Kesselring1, Irma Querques2,3
1Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen 63225, Germany.
Nucleic Acids Research
|February 21, 2022
Summary
Researchers modified the Sleeping Beauty (SB) transposase to improve genome engineering safety. Mutants show increased target specificity, directing integrations away from harmful genomic areas, enhancing its gene therapy potential.
Area of Science:
- Molecular Biology
- Genomics
- Gene Therapy
Background:
- The Sleeping Beauty (SB) transposon system is a widely used tool for genome engineering.
- Random integration of SB transposons poses genotoxic risks in therapeutic applications.
Purpose of the Study:
- To investigate the role of specific amino acids (H187, P247, K248) in the SB transposase's target site selection.
- To engineer SB transposase variants with altered and potentially safer integration profiles.
Main Methods:
- Structural modeling to identify key amino acid residues.
- Saturation mutagenesis of selected residues in the SB transposase.
- Transposon integration profiling to analyze target site selection of mutant variants.
Main Results:
- Mutagenesis yielded SB transposase variants with altered target site selection.
- Specific mutants (H187V, K248R) showed increased integration specificity into palindromic AT repeats.
- These variants redirected integrations away from exons, regulatory elements, and nucleosomal DNA.
Conclusions:
- Amino acids H187, P247, and K248 are critical for SB transposase target site selection.
- Engineered SB variants demonstrate enhanced specificity and safety for gene therapy applications.
More Related Videos
10:58Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma
Published on: February 22, 2015
13.1K
07:04Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System
Published on: February 4, 2021
2.2K
Related Concept Videos
02:57DNA-only Transposons
14.9K
DNA-only transposons are called autonomous transposons since they code for the enzyme transposase that is required for the transposition mechanism. Insertion of transposons can alter gene functions in multiple ways. They can mutate the gene, alter gene expression by introducing a novel promoter or insulator sequence, introduce new splice sites, and change the mRNA transcripts produced, or remodel chromatin structure.
The donor site from where the transposon is excised is either degraded or...
The donor site from where the transposon is excised is either degraded or...
14.9K
02:13Overview of Transposition and Recombination
16.3K
Transposons make up a significant part of genomes of various organisms. Therefore, it is believed that transposition played a major evolutionary role in speciation by changing genome sizes and modifying gene expression patterns. For example, in bacteria, transposition can lead to conferring antibiotic resistance. Movement of transposable elements within the genetic pool of pathogenic bacteria can aid in transfer of antibiotic-resistant genetic elements. In eukaryotes, transposons can carry out...
16.3K
01:24Transposons
245
Transposons, or "jumping genes," are small mobile genetic elements (MGEs) that range from 700 to 40,000 base pairs in length. They are found in all organisms and can move within the same chromosome or transfer to different chromosomes. In some cases, transposons can also jump between different host DNA molecules, such as plasmids or viruses, contributing to genetic variability.Barbara McClintock first discovered these mobile genetic elements in the 1940s while studying maize genetics, and she...
245
02:53Conservative Site-specific Recombination and Phase Variation
6.2K
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
6.2K
03:18Non-LTR Retrotransposons
12.0K
As the name suggests, non-LTR retrotransposons lack the long terminal repeats characteristic of the LTR retrotransposons. Additionally, both LTR and non-LTR retrotransposons use distinct mechanisms of mobilization. Non-LTR retrotransposons are further divided into two classes - Long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), both of which occur abundantly in most mammals, including humans. Some of the active non-LTR retrotransposons in humans are L1...
12.0K
02:57piRNA - Piwi-interacting RNAs
7.1K
PIWI-interacting RNAs, or piRNAs, are the most abundant short non-coding RNAs. More than 20,000 genes have been found in humans that code for piRNAs while only 2000 genes have been found for miRNAs. piRNAs can act at the transcriptional and post-transcriptional levels and have a vital role in silencing transposable elements present in germ cells. They are also involved in epigenetic silencing and activation. Previously, they were thought to function only in germ cells but new evidence suggests...
7.1K