An Engineered Cas-Transposon System for Programmable and Site-Directed DNA Transpositions

Abstract

Efficient site-directed insertion of heterologous DNA into a genome remains an outstanding challenge. Recombinases that can integrate kilobase-sized DNA constructs are difficult to reprogram to user-defined loci, while genomic insertion using CRISPR-Cas methods relies on inefficient host DNA repair machinery. Here, we describe a Cas-Transposon (CasTn) system for genomic insertions that uses a Himar1 transposase fused to a catalytically dead dCas9 nuclease to mediate programmable, site-directed transposition. Using cell-free in vitro assays, we demonstrated that the Himar-dCas9 fusion protein increased the frequency of transposon insertion at a single targeted TA dinucleotide by >300-fold compared to a random transposase, and that site-directed transposition is dependent on target choice while robust to log-fold variations in protein and DNA concentrations. We also showed that Himar-dCas9 mediates directed transposition into plasmids in Escherichia coli. This work highlights CasTn as a new modality for host-independent, programmable, site-directed DNA insertions.

The Versatile Type V CRISPR Effectors and Their Application Prospects

“…The DSB induced by the sequence-specific nucleases at the target sites are fatal in organisms without effective NHEJ or HDR (Xu et al, 2015 ). To overcome this limitation, researchers attempted to connect different transposases to catalytically inactivated dCas9 (site-specific mutations were introduced into the HNH and RuvC domains to abrogate the cleavage activity without affecting the targeted binding effect) to obtain a series of dCas9–transposase fusion proteins [such as dCas9- Himar1 (Chen and Wang, 2019 ), dCas9- piggyBac (Hew et al, 2019 ), and dCas9- Sleeping Beauty (Kovač et al, 2020 )]. These fusions limit the random insertion of transposase, so that transposition occurs only near the point where dCas9 is targeted.…”

“…Although the transposition reaction does not depend on the DSB repair ability of the host, limitations of the fusion protein by the PAM of dCas9 and the transposase insertion hot spot make this tool less flexible. For example, dCas9-Himar1 requires a TA dinucleotide within 15 bp behind the 5′ end of the gRNA (Chen and Wang, 2019 ).…”

Advances and Obstacles in Homology-Mediated Gene Editing of Hematopoietic Stem Cells

“…As summarized in this section, various cellular nucleases have been engineered to recognize individual target sequences and induce the necessary DSBs and DNA repair response for targeted DNA modification ( Figure 1 c). Alternative strategies to manipulate cellular genomes that do not rely on double-stranded DNA cleavage, including base editors [ 8 , 9 , 10 , 11 ], prime editors [ 12 ], and transposases/recombinases [ 13 , 14 , 15 , 16 ], were also developed in recent years and have been reviewed elsewhere [ 17 ].…”

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

“…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 ]. They demonstrated that the Himar–dCas9 fusion protein improved the frequency of transposon insertion at a single targeted TA dinucleotide by > 300-fold compared to the un-fused transposase.…”

“…They demonstrated that the Himar–dCas9 fusion protein improved the frequency of transposon insertion at a single targeted TA dinucleotide by > 300-fold compared to the un-fused transposase. This work highlights CasTn as a new modality for host-independent, programmable and site-directed DNA insertions[ 87 ].…”