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

Abstract

Various methods have been used in targeted gene knock-in applications. CRISPR-based knock-in strategies based on homology-independent repair pathways such as CRISPR HITI have been shown to possess the best efficiency for gene knock-in in mammalian cells. However, these methods suffer from the probability of plasmid backbone insertion at the target site. On the other hand, studies trying to combine the targeting ability of the Cas9 molecule and the excision/integration capacity of the PB transposase have shown random integrations. In this study, we introduce a new homology-independent knock-in strategy, Transposase-CRISPR mediated Targeted Integration (TransCRISTI), that exploits a fusion of Cas9 nuclease and a double mutant piggyBac transposase. In isogenic mammalian cell lines, we show that the TransCRISTI method demonstrates higher efficiency (72%) for site-specific insertions than the CRISPR HITI (44%) strategy. Application of the TransCRISTI method resulted in site-directed integration in 4.13% and 3.69% of the initially transfected population in the human AAVS1and PML loci, respectively, while the CRISPR HITI strategy resulted in site-directed integration in the PML locus in only 0.6% of cells. We also observed lower off-target and random insertions in the TransCRISTI group than the CRISPR HITI group. The TransCRISTI technology represents a great potential for the accurate and high-efficiency knock-in of the desired transposable elements into the predetermined genomic locations.

Precise genetic engineering with <i>piggyBac</i> transposon in plants

“…It has been reported that PBase mutants showing higher transposase activity compared with hyPBase were developed using a screening system in yeast and applied to mammalian cells (Wen et al 2020). Furthermore, the fusion of PBase and TALE or CRISPR/Cas9 has been reported to enable the targeted integration and high-efficiency knock-in into a target gene in mammalian cells (Owens et al 2013;Rezazade Bazaz et al 2022). Recently, in mammalian cells, especially in hard-to-transfect cells like human iPSC, the piggyBac-mediated transgenesis system has made it possible to provide an effective gene correction approach using Prime Editor, which enables precise gene editing with the reverse transcription (Eggenschwiler et al 2021;Wolff et al 2021).…”

biGMamAct: efficient CRISPR/Cas9-mediated docking of large functional DNA cargoes at theACTBlocus

“…For making our method the more universal and as efficient as possible we decided to rely on NHEJ-repair mechanisms and to involve the powerful and underrated Homology-Independent Targeted Insertion (HITI) 27 . Indeed, HITI has been reported to be the most efficient strategy to mediate knock-ins 27,44 . In addition, and in contrast to low efficiency HDR, HITI was shown to occur at every stage of cellular life, rendering our method applicable into dividing and non-dividing cells.…”