In vitro transposon targeting using a catalytically inactive Cas9

TLDR: In this paper, the Hsmar1 transposase was fused to a catalytically inactive Cas9 to bias transposon insertions into the vicinity of the target site bound by a guide RNA-dCas9 ribonucleoprotein complex.

Abstract: Transposases are attractive tools for the integration of therapeutic transgenes into the chromosome for gene therapy applications. Typically, transgenes can be flanked with inverted-terminal repeat sequences, which are recognised by the transposase and integrated at random sites. Minimising detrimental insertions of transgenes is a key goal in the development of gene delivery vectors for gene therapy. We fused the Hsmar1 transposase to a catalytically inactive Cas9. Our aim was to bias transposon insertions into the vicinity of the target site bound by a guide RNA-dCas9 ribonucleoprotein complex. Although we could not detect any targeted transposition events in vivo, we achieved a 15-fold enrichment of transposon insertions into a 600-bp target site in an in vitro plasmid-to-plasmid assay. Additionally, we show that among those integrations that were successfully targeted, the location is tightly constrained to a site immediately to one side of the guide RNA target site. We present an in vitro proof-of-concept study demonstrating that the transposase insertion profile can be biased using a catalytically inactive Cas9 variant as a programmable DNA-binding module. One factor that limits the utility of this approach is that the transposon continues to integrate randomly. Although the dCas9 domain can be targeted to chromosomal lacZ, as evidenced by transcriptional repression, we were unable to detect any targeted insertions in the vicinity of the target site. Any targeted insertions that did occur were masked be a much larger number of random insertions. It is therefore necessary to develop a method for the temporal control of the transposase to allow Cas9 time to locate its target site.