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.
Highlights
Targeted gene knock-in methods have been used in a wide range of applications such as gene therapy, gene correction, or the study of gene function[1]
A new plasmid is generated by the Cas[9].PBdm-mediated integration of the donor transposon upstream of the promoterless reporter in HEK293T cells leading to the expression of punctate PML nuclear bodies with red fluorescence. (B) The left panel is the fluorescent image of DsRed2-PML4 expressing cells 48 h post-transfection
The results showed that there were 3.35-fold higher interplasmid events in the fused effector group than those in the group with separate effectors (p < 0.05; 4.13% vs. 1.23% DsRed2-PML4 positive cells, respectively) (Fig. 1C)
Summary
Targeted gene knock-in methods have been used in a wide range of applications such as gene therapy, gene correction, or the study of gene function[1]. The donor in the HITI method is simple to produce and does not need the cloning of the flanking homology arms, synthesis of large ssDNA donor fragments, or any other modifications In this strategy, the sgRNA binding site(s) are placed at one or either side of the gene of interest (GOI) cassette. The sgRNA binding site(s) are placed at one or either side of the gene of interest (GOI) cassette These sites on the donor molecule when targeted by Cas[9] (CRISPR-associated protein 9), provide a linear cassette that will be integrated into the genome. The DsRed2-PML4 expressing cells are considered as positive cells for the integration of the donor fragment (containing CMV promoter flanked by PB transposon ITRs) into the sgRNA #1 binding site (sg BS #1) upstream of the promoterless acceptor construct. The strategy of dCas[9].PB using dual sgRNAs similar to the previously published methods suffered from off-target and random integrations
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