Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection

Abstract

Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing.

High-efficiency nuclear transformation of the microalgae Nannochloropsis oceanica using Tn5 Transposome for the generation of altered lipid accumulation phenotypes

“…The use of the transposon resulted in transformation efficiencies (1.57 × 10 −2 transformants per μg of DNA) much higher than those described for other types of microalgae [42–46], with a potential number of total transformants of 2,700,400 mutant clones per microgram of DNA. Importantly, nuclear transformation using endogenous promoters in Nannochloropsis strains has resulted in lower transformation efficiencies of around 1.25–0.6 × 10 −06 [42, 43].…”

Targeted DNA transposition in vitro using a dCas9-transposase fusion protein

“…The single biggest problem in targeted-transposition protocols is that successful targeting in a particular cell is usually accompanied by random integrations. It should be possible to minimize this problem by delivering the transpososome to the cells after in vitro assembly (29). However, to overcome random integration events, it will be necessary to control the timing of integration to allow an opportunity for the targeting moiety to find its binding site.…”

“…Furthermore, if mariner transpososomes are assembled in vitro , they can be delivered to cells directly [Ref. (29) and our unpublished experiment). Hsmar1 transposase also has much lower non-specific nuclease activity than other mariner transposases such as Mos1, Mboumar1 and Himar1 (30–35).…”

Toward a genetic system in the marine cyanobacterium Prochlorococcus

“…We are confident, however, that effective solutions can be found to overcome these hurdles. Solutions might come from new transposome systems [ 86 ], which could reveal more efficient means than the Tn5 transposition; from the delivery of Cpf1-RNA complexes inside cells [ 87, 88 ]; from using more systematic approaches to evade restriction-modification defences [ 15 ]; through the discovery of yet unknown defence systems [ 89 ] that are hampering transformation; or finally by harnessing endogenous mobile genetic elements, which have been tailored by evolution to work efficiently in these minimal cells. The latter is the direction we are currently exploring.…”