A single amino acid switch converts the Sleeping Beauty transposase into an efficient unidirectional excisionase with utility in stem cell reprogramming

Abstract

The Sleeping Beauty (SB) transposon is an advanced tool for genetic engineering and a useful model to investigate cut-and-paste DNA transposition in vertebrate cells. Here, we identify novel SB transposase mutants that display efficient and canonical excision but practically unmeasurable genomic re-integration. Based on phylogenetic analyses, we establish compensating amino acid replacements that fully rescue the integration defect of these mutants, suggesting epistasis between these amino acid residues. We further show that the transposons excised by the exc+/int− transposase mutants form extrachromosomal circles that cannot undergo a further round of transposition, thereby representing dead-end products of the excision reaction. Finally, we demonstrate the utility of the exc+/int− transposase in cassette removal for the generation of reprogramming factor-free induced pluripotent stem cells. Lack of genomic integration and formation of transposon circles following excision is reminiscent of signal sequence removal during V(D)J recombination, and implies that cut-and-paste DNA transposition can be converted to a unidirectional process by a single amino acid change.

piggyBac-Based Non-Viral In Vivo Gene Delivery Useful for Production of Genetically Modified Animals and Organs

“…Li et al [21] developed a novel PB mutant having an excision activity but not a genome reintegration capability, which is now called "excision-only PB transposase". A similar observation was also reported by Kesselring et al [22], who constructed an "excision only" SB transposase through a single amino acid switch in the SB transposase. Thus, DNA transposons, as exemplified by PB, are considered to be promising non-viral alternatives.…”

Jumping Ahead with Sleeping Beauty: Mechanistic Insights into Cut-and-Paste Transposition

“…An interesting variant of the SB transposase which has recently been described is the K248T mutant [ 108 ]. This mutant is competent in transposon excision but has the feature of deficiency in transposon integration [ 108 ]. After excision by K248T, extrachromosomal transposon circles are formed which apparently cannot undergo integration.…”

Potential of transposon-mediated cellular reprogramming towards cell-based therapies

“…These sequence-specific integrations offer significant advantages over traditional virus-based integrating vectors by avoiding insertion into unwanted regions[ 90 - 93 ]. Another approach applied to generate “transient transgenesis” by mutation at position 248 in the SB transposase to gain further insight into the transposition mechanism and for the generation of reprogramming factor-free iPS cells[ 17 ]. The amino acid present at position 248 of the SB transposase is involved in an interaction with target DNA, and because of the absence of integration activity, transposon removal by these transposase mutants results in extra-chromosomal circles, thereby terminating the transposition reaction[ 17 , 94 ].…”

“…Another approach applied to generate “transient transgenesis” by mutation at position 248 in the SB transposase to gain further insight into the transposition mechanism and for the generation of reprogramming factor-free iPS cells[ 17 ]. The amino acid present at position 248 of the SB transposase is involved in an interaction with target DNA, and because of the absence of integration activity, transposon removal by these transposase mutants results in extra-chromosomal circles, thereby terminating the transposition reaction[ 17 , 94 ]. This indicates that by the switching of a single amino acid, the SB transposase has into efficient unidirectional removal ability with utility in cellular reprogramming.…”

“…In this respect, Sleeping Beauty (SB) and piggyBac (PB) transposon systems appear as attractive tools for somatic cell reprogramming due to their efficient gene delivery and their ability to be excised from the cells after reprogramming, which helps overcome the limitations of viral-based reprogramming technologies. Transposon systems have a number of additional advantages, such as (1) Cargo capacity of up to 100 kb[ 14 , 15 ]; (2) No bias to integrate in expressed genes or promoter regions; (3) Possibility of seamless removal of the transposon[ 16 , 17 ]; (4) Cost-effective production of the basic plasmids; (5) Reduced innate immunogenicity; and (6) No requirement for a specialized biosafety facility.…”