Development of Hsmar1 transposon tools for research and biotechnologyTools Bhatt, Shivam (2019) Development of Hsmar1 transposon tools for research and biotechnology. PhD thesis, University of Nottingham.
AbstractDNA transposons are mobile genetic elements that relocate between genomic loci. This relocation is termed transposition and is catalysed by an element-encoded enzyme called transposase. Hsmar1 is a DNA transposon that invaded the primate genome ~50 Mya and was active for ~13 My. Over the last decade, the molecular mechanisms of Hsmar1 transposition and autoregulation have been covered extensively. I took advantage of this mechanistic understanding to develop transposon technologies using Hsmar1 as a proof-of-concept platform. In research and biotechnology, transposons are used for insertional mutagenesis, transgenesis, gene therapy and next-generation sequencing. To improve existing transposon tools, I sought to establish increased control over the transposition reaction. Most transposases integrate their transposons semi-randomly throughout the genome. To control where transposons are integrated, I fused the Hsmar1 transposase to a catalytically inactive Cas9 variant, dCas9. The aim was to bias transposon insertions into the vicinity of a pre-determined target site bound by a guide RNA-dCas9-transposase ribonucleoprotein complex. I detected a 14.8-fold enrichment of transposon insertions into a 600 bp target site in an in vitro plasmid-to-plasmid assay compared to unfused transposase. Additionally, I show that 100 % of targeted insertions occur within 22 bp to one side of the guide RNA binding site. Next, I developed a light-inducible transposase for non-invasive, spatio-temporal control over the onset of transposition. Light-activated transposase reached up to 50 % of the activity of wild-type. To find hyperactive transposases, I constructed a library of 2 x 105 transposase mutants. I isolated three hyperactive variants and found that hyperactivity occurs due to faster transposon-end synapsis. Lastly, I developed an Hsmar1 transpososome, which is a pre-assembled synaptic complex in vitro. I show that this complex can be chemically transformed into E. coli for one-step insertional mutagenesis. Furthermore, the transpososome can also be lipofected into human cells, where the level of transposition exceeds that of the traditionally used transposase-helper plasmids.
Actions (Archive Staff Only)
|
Tools
Tools
![[img]](/style/images/fileicons/application_pdf.png)
