Applications of TALENs and CRISPR/Cas9 in Human Cells and Their Potentials for Gene Therapy

Abstract

The newly developed TALENs and emerging CRISPR/Cas9 have spurred interests in the field of genome engineering because of their ease of customization and high-efficient site-specific cleavages. Although these novel technologies have been successfully used in many types of cells, it is of great importance to apply them in human-derived cells to further observe and evaluate their clinical potentials in gene therapy. Here, we review the working mechanism of TALEN and CRISPR/Cas9, their effectiveness and specificity in human cells, and current methods to enhance efficiency and reduce off-target effects. Besides, CCR5 gene was chosen as a target example to illustrate their clinical potentials. Finally, some questions are raised for future research and for researchers to consider when making a proper choice bases on different purposes.

A CRISPR view of development

“…The ability to introduce deletions as well as point mutations in a wide variety of cell types allows for the generation of cell lines with human disease-causing mutations. A large number of genes in a diversity of human cell types have already been modified (for review, see Niu et al 2014a), providing the capacity to study mechanistic effects of these mutations. The relative ease of generating mutant animals will yield many additional animal models of disease and supply a means of testing whether specific polymorphisms are the proximal cause of disease in vivo.…”

To Crispr And Beyond: The Evolution Of Genome Editing In Stem Cells

“…TALENs-mediated gene correction has been demonstrated in fibroblasts derived from epidermolysis bullosa patients [63]. Additionally, numerous studies have used TALENs on human pluripotent stem cells for development of targeted gene therapy [64][65][66][67].…”

Modelling Neurodegenerative Diseases Using Human Pluripotent Stem Cells

“…These are composed of a sequence-specific DNA-binding domain and a non-specific DNA cleavage module [18]. The DNA binding domain contains a series of tandem repeats comprising 33-35 amino acids, similar to tandem repeats first discovered in the plant pathogen Xanthomonas [19].…”

“…This method consists of a specialized two-RNA structure containing CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA), which are able to bind as a monomer to DNA strands next to a protospacer adjacent motif (PAM) composed of the sequence, 5'NGG'3 [18]. This chimeric RNA is known as a guide RNA (gRNA) and facilitates a DSB by guiding an endonuclease (first derived from Streptococcus pyogenes (Cas9)) to induce cleavage near the PAM site (Figure 1).…”

“…Another potential disadvantage is the potential increased off-target binding and cleavage compared to TALENs, due to its monomeric action, shorter target sequences and greater chance of binding to identical target sites elsewhere in the genome [9,23]. Like ZFNs, nickases have been effectively used to prevent off-target binding and cleavage [18].…”