Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells

Abstract

The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 ‘safe harbor’ locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform.

Optical control of mammalian endogenous transcription and epigenetic states

“…The ssDNA-based genome of AAV is less susceptible to recombination, thus providing an advantage over RNA-based lentiviral vectors 27 for the packaging and delivery of highly repetitive TALE sequences. 293FT cells (Life Technologies) were grown in antibiotic-free D10 media (DMEM high glucose with GlutaMax and Sodium Pyruvate, 10% heatinactivated Hyclone FBS, and 1% 1M HEPES) and passaged daily at 1:2-2.5.…”

Genome-Editing Technologies: Principles and Applications

“…Second, TALENs have been reported to show improved specificity and reduced toxicity compared to some ZFNs (Mussolino et al 2014), potentially because of their increased affinity for target DNA or perhaps a greater energetic penalty for associating with base mismatches. However, TALENs are substantially larger than ZFNs, and have a highly repetitive structure, making their efficient delivery into cells through the use of lentivirus (Holkers et al 2013) or a single adeno-associated virus (AAV) particle challenging. Methods for overcoming these limitations have emerged as TALENs can be readily delivered into cells as mRNA (Mahiny et al 2015;Mock et al 2015) and even protein Liu et al 2014a), although alternative codon usage and amino acid degeneracy can also be leveraged to express RVD arrays that might be less susceptible to recombination (Kim et al 2013a).…”

CRISPRs encounter Cocktail: a dawn for curing HIV/AIDS

“…ZFNs are discovered in 1996 [11] and then widely used to manipulate the genomes since they can recognize specific DNA domains and cleave them with double-strain breaks(DSBs). TALENs are similar to ZFNs in working mechanisms, but TALENs behave better in specificity to DNA domains [12]. However, both ZFNs and TALENs were limited to be applied intensively because of the difficulty in their construction and susceptibility to induce genomic rearrangements [13].…”