CRISPR/dCas9‐mediated transposition with specificity and efficiency of site‐directed genomic insertions

Abstract

The ability and efficiency of targeted nucleases to perform sequence replacements or insertions into the genome are limited. This limited efficiency for sequence replacements or insertions can be explained by the dependency on DNA repair pathways, the possibility of cellular toxicity, and unwanted activation of proto-oncogenes. The piggyBac (PB) transposase uses a very efficient enzymatic mechanism to integrate DNA fragments into the genome in a random manner. In this study, we fused an RNA-guided catalytically inactive Cas9 (dCas9) to the PB transposase and used dual sgRNAs to localize this molecule to specific genomic targets. We designed and used a promoter/reporter complementation assay to register and recover cells harboringspecific integrations, where only by complementation upon correct genomic integration, the reporter can be activated. Using an RNA-guided piggyBac transposase and dual sgRNAs, we were able to achieve site-directed integrations in the human ROSA26 safe harbor region in 0.32% of cells. These findings show that the methodology used in this study can be used for targeting genomic regions. An application for this finding could be in cancer cells to insert sequences into specific target regions that are intended to be destroyed, or to place promoter cargos behind the tumor suppressor genes to activate them. K E Y W O R D SCRISPR-associated protein 9, gene insertion, Insertional mutagenesis, piggyBac transposase, transposases 2 of | GOSHAYESHI Et Al.

Transposase-CRISPR mediated targeted integration (TransCRISTI) in the human genome

“…In a recent study, the fusion of SB to the dCas9 increased the probability of insertion to a 300-bp window nearby the target site which was still far from a precise site-specific insertion event 24 . We have recently shown that by using dCas9 fused to PB and dual sgRNAs, site‐directed integrations could be achieved in 0.32% of transfected cells 14 . However, in all of the above-mentioned studies, the usefulness of these chimeric molecules for gene therapy applications is largely affected by random genomic integrations, inherent to the activity of transposase.…”

“…Several previously published articles have used a similar approach to determine the efficiency of site-specific insertions 10 , 13 , 20 , 25 . In the second approach similar to our previous report 14 , we used the promoter/reporter complementation assay to register and recover cells harboring specific integrations. In this strategy, the promoterless reporter fragments can only be activated (by the endogenous promoters) when they are placed precisely in the integration site.…”

“…Another study showed that the excision competent/integration defective (Exc + Int − ) hyperactive piggyBac transposase 11 , 12 fused to dCas9 was able to target the CCR5 safe harbor sequence, albeit inefficiently 13 . We have recently shown that dCas9.PB using dual sgRNAs can achieve site-directed transposition in 0.32% of cells out of the initially transfected population 14 . In that study, we designed and used a promoter/reporter complementation assay to register cells with specific integrations, where only by complementation upon correct genomic integration, the reporter is activated.…”

Applications of piggyBac Transposons for Genome Manipulation in Stem Cells

“…A specific Cas9 mutant lacking endonuclease activity (dCas9) fused with transcriptional repressor or activation domains has also been generated to promote transcriptional inhibition or activation when coexpressed with targeted sgRNAs [ 85 , 86 ]. Lena et al fused dCas9 to PBase and targeted it to specific genomic sites using dual sgRNAs [ 87 ]. Thus, the ease of design and application of dCas9-PBase, which can edit genes at precise genomic loci, improves future medical applications.…”

Research progress in gene editing technology

“…Although not yet studied outside of bacteria, CAST still holds promise for a next-generation gene editing system with enhanced efficiency [116][117][118][119].…”