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

Abstract

Homology-directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Here we present an in vitro mechanistic study that demonstrates efficient Cas9 targeting of the mariner transposon Hsmar1. Integrations were unidirectional and tightly constrained to one side of the sgRNA binding site. Further analysis of the nucleoprotein intermediates demonstrated that the transposase and Cas9 moieties can bind their respective substrates independently or in concert. Kinetic analysis of the reaction in the presence of the Cas9 target–DNA revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase moiety behaved normally and was proficient for integration in vitro and in Escherichia coli. Specific integration into the lacZ gene in E. coli was obscured by a high background of random integrations. Nevertheless, Cas9 is an attractive candidate for transposon-targeting because it has a high affinity and long dwell-time at its target site. This will facilitate a future optogenetic strategy for the temporal control of integration, which will increase the ratio of targeted to untargeted events.

RNA-guided Retargeting of Sleeping Beauty Transposition in Human Cells

“…We further detect an asymmetric distribution of insertions around the target site. Asymmetric distributions have been found in several previous targeting studies, for example a study using the ISY100 transposon (which, like SB, is a member of the Tc1/mariner transposon superfamily) in combination with the ZF domain Zif268 in E. coli (76) or in vitro experiments with dCas9/Hsmar1 fusions (55). Enrichment mainly occurring downstream of the sgRNA target site was somewhat surprising, as domains fused to the Cterminus of Cas9 are expected to be localized closer to the 5'-end of the target strand (77), or upstream of the sgRNA binding site.…”

“…Using dCas9 as a targeting domain for a transposon could combine this great flexibility with the advantages of integrating vectors. By using the Hsmar1 human transposon (54), a 15-fold enrichment of transposon insertions into a 600-bp target region was observed in an in vitro plasmid-to-plasmid assay employing a dCas9-transposase fusion (55). However, no targeted transposition was detected with this system in bacterial cells.…”

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

“…Surprisingly, the dCas9-PB chimera protected it from insertions instead of targeting the HPRT locus. Although, PB is considered to be the most efficient system for gene delivery in vivo [ 84 , 85 ], it impedes the development of advanced applications such as direct delivery of transposons[ 86 ]. To resolve this difficulty, Chen and Wang described a Cas-Transposon (CasTn) system for genomic insertions which uses a Himar1 transposase fused with a dCas9 nuclease to mediate programmable, site-directed transposition[ 87 ].…”

Integration of diverse DNA substrates by a casposase can be targeted to R-loops in vitro by its fusion to Cas9

“…The effectiveness of HR for genetic editing is offset by drawbacks including the triggering of unpredictable genome re-arrangements and low efficacy. Other strategies have sought to overcome the problems of HR-dependent genetic insertion by exploiting interactions or fusions of Cas9 with transposases (22)(23)(24)(25), and by repurposing group-II introns (26). We were interested in casposases for this purpose because of their ability to integrate a wide variety of DNA substrates.…”