Abstract
Recently, we proved that Sleeping Beauty (SB) transposon integrates into non-TA sites at a lower frequency. Here, we performed a further study on the non-TA integration of SB and showed that (1) SB can integrate into non-TA sites in HEK293T cells as well as in mouse cell lines; (2) Both the hyperactive transposase SB100X and the traditional SB11 catalyze integrations at non-TA sites; (3) The consensus sequence of the non-TA target sites only occurs at the opposite side of the sequenced junction between the transposon end and the genomic sequences, indicating that the integrations at non-TA sites are mainly aberrant integrations; and (4) The consensus sequence of the non-TA target sites is corresponding to the transposon end sequence. The consensus sequences changed following the changes of the transposon ends. This result indicated that the interaction between the SB transposon end and genomic DNA (gDNA) may be involved in the target site selection of the SB integrations at non-TA sites.
Highlights
Sleeping Beauty (SB) transposon, a member of the Tc1/mariner family (Ivics et al, 1997), is the most widely used transposon genetic tool for gene therapy and the generation of genomewide mutations (Dupuy et al, 2005; Starr et al, 2009; O’Donnell et al, 2012; Guo et al, 2016)
Does the traditional SB11 transposase catalyze non-TA integration too? (3) Why does this consensus sequence only occur at one side of the integration site? and (4) We found that the consensus sequence flanking the integration site is the same as the sequence of the transposon ends, which was speculated the result of the interaction between the transposase and the target site, but is it possible that this phenomenon is the result of the interaction between the transposon end and the target
We found non-TA integrations in the co-transfection of both SB100X and SB11 plasmids, indicating that SB11 can mediate integrations at non-TA sites as well as SB100X
Summary
Sleeping Beauty (SB) transposon, a member of the Tc1/mariner family (Ivics et al, 1997), is the most widely used transposon genetic tool for gene therapy and the generation of genomewide mutations (Dupuy et al, 2005; Starr et al, 2009; O’Donnell et al, 2012; Guo et al, 2016). It was thought that SB, as well as other Tc1/mariner transposons, strictly integrates into TA dinucleotides (Ivics et al, 1997; Plasterk et al, 1999; Yant et al, 2005). This conclusion was based on the limited integration data before generation sequencing (NGS) was widely used. We analyzed more than 2 million SB integration sites in mouse BaF3 cells and proved that SB could integrate into non-TA sites at a frequency of ~1.4% (Guo et al, 2018). While reporting the non-canonical integration of SB for the first time, our study raised several new questions: (1) given the integrations at non-TA sites were found in mouse cell lines, are there integration at non-TA sites in human cell lines? (2) The non-TA integrations we found were mediated by the hyperactive transposase version, SB100X (Mátés et al, 2009)
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