Progress and Problems with Viral Vectors for Delivery of Talens

Abstract

Transcription Activator-Like Effector Nucleases (TALENS) and Their Mode of ActionFor sequence-specific genome engineering and its biotechnological and medical applications various systems were tested. Most recent tools include designer nucleases and the bacteria derived clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system using short RNA to induce precise cleavage at endogenous genomic loci [1,2]. Designer nucleases are mainly represented by zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) [3][4][5][6][7], combining features of a customized DNA binding motif for sequence-specific DNA binding domain and a nuclease for introduction of doubled-strand DNA (dsDNA) breaks at the target site.TALENs are a promising new class of designer nucleases that can be specifically designed to bind DNA sequences of interest and to introduce dsDNA breaks. They are chimeric proteins consisting of a N-terminal nuclear localization signal, a central DNA binding domain and a C-terminal FokI nuclease domain. The DNA binding domain originates from transcription activator like effectors (TALEs) of the bacterial plant pathogen Xanthomonas, used by this bacterium to alter the expression of several host genes [8]. Its main characteristic is a central repeat region consisting of a variable number of incomplete tandem repeats that are usually comprised of 33-35 amino acids (aa) that are identical except for the hypervariable repeat-variable diresidue (RVD) at a positions 12 and 13 [6]. With some degree of degeneracy, the RVD of each repeat is specific for binding a corresponding nucleotide in their contiguous target DNA sequence [6,8]. The C-terminal FokI domain provides non-specific endonuclease activity and similar to ZFNs, binding of the TALE domains upstream and downstream of the respective DNA target and subsequent dimerization at the FokI nuclease domain lead to dsDNA breaks. It is of note that the spacer between the TALEN DNA binding sites needs to be considered when designing novel TALENs [6,7]. After binding, dsDNA breaks are introduced which can than activate cellular pathways either leading to homologous recombination in the presence of the respective homologous donor AbstractIt has long been envisaged that gene disruption or gene correction in affected target cells can be efficiently conducted in vitro and in vivo and over the recent years several tools for achieving this goal were developed. Designer nucleases such as zinc finger nucleases (ZFNs) were extensively explored and more recently transcription activator-like effector nucleases (TALENs) were introduced for sequence-specific genome engineering in the mammalian genome. ZFNs and TALENs are fusion proteins containing a customized DNA-binding motif for sequence-specific DNA binding linked to a nuclease for introduction of double-stranded DNA breaks. Both systems were explored in mammalian cells using non-viral and viral delivery methods. Herein, we will provide a state-ofthe-art overview of available virus-base...

Creating cell and animal models of human disease by genome editing using CRISPR/Cas9

“…31 In other words, TALE repeats can target longer sequences of interest. 32 33 is noteworthy. Overall, the aforementioned problems limit the applications of TALENs, and further research is required to resolve these issues ( Table 1).…”

“…Previous studies conducted with the aim of overcoming these problems revealed that the number of repeats has a direct relationship with specificity in TALENs and, in addition, attempts to join two TALEN expression cassettes with the aim of improving efficiency using vector systems such as adeno‐associated virus (AAV), lentivirus and adenovirus would be unsuccessful. Of the efforts made to resolve this issue, the use of internal ribosomal entry sites (IRES) or A2 peptides in the design of vectors is noteworthy. Overall, the aforementioned problems limit the applications of TALENs, and further research is required to resolve these issues (Table ).…”

Pluripotent stem cell applications for regenerative medicine

“…TALENs function similarly to ZFN, but possess dramatically improved specificity. There have been several reports demonstrating successful applications in hESCs/hiPSCs [54–56], however, TALENs also are subjected to the same concerns as ZFNs in that their delivery is mostly dependent on viral transduction [57], there is a high rate of non-specific targeting that may result in tumorigenesis (even if the intended target is a safe harbor site) [58], and validation can be laborious.…”

Balance between DNA‐binding affinity and specificity enables selective recognition of longer target sequences in vivo

“…Because the size of the AAV vector genome is limited to less than ~4.7 kb, it is difficult for the vector to hold the spCas9 or TALEN gene. Consequently, two viruses are required for TALEN delivery, one for the right part and one for a left part of TALEN . In addition to the delivery of the intact enzymes, introduction of control elements to customize expression in the system is also being considered.…”

“…Consequently, two viruses are required for TALEN delivery, one for the right part and one for a left part of TALEN. 8 In addition to the delivery of the intact enzymes, introduction of control elements to customize expression in the system is also being considered. This makes the physical size limit an even more serious problem.…”