Efficient TALEN construction and evaluation methods for human cell and animal applications

Abstract

Transcription activator-like effector nucleases (TALENs) have recently arisen as effective tools for targeted genome engineering. Here, we report streamlined methods for the construction and evaluation of TALENs based on the 'Golden Gate TALEN and TAL Effector Kit' (Addgene). We diminished array vector requirements and increased assembly rates using sixmodule concatemerization. We altered the architecture of the native TALEN protein to increase nuclease activity and replaced the final destination vector with a mammalian expression/ in vitro transcription vector bearing both CMV and T7 promoters. Using our methods, the whole process, from initiating construction to completing evaluation directly in mammalian cells, requires only 1 week. Furthermore, TALENs constructed in this manner may be directly applied to transfection of cultured cells or mRNA synthesis for use in animals and embryos. In this article, we show genomic modification of HEK293T cells, human induced pluripotent stem cells, Drosophila melanogaster, Danio rerio and Xenopus laevis, using custom-made TALENs constructed and evaluated with our protocol. Our methods are more time efficient compared with conventional yeast-based evaluation methods and provide a more accessible and effective protocol for the application of TALENs in various model organisms.

Genome Editing Tools for Functional Analysis of HNF1A and IL6ST Genes

“…[7] The assembly of two IL6ST TALE repeats was conducted using a modified protocol involving intermediary 6-module pFUS array vectors. [8] These modified pFUS vectors can reduce the number of module plasmids and improve the success rate of Golden Gate assembly. The additional plasmid kit used for building TALENs and TALE-TFs was a gift from Takashi Yamamoto (Addgene kit # 1000000030).…”

“…The pair of TALE nucleases (TALENs) [7] introduces double-stranded breaks at the targeted site in a manner similar to ZFNs, but with the advantages of highly predictable modular assembly and the possibility of targeting almost any DNA sequence. Plasmid kits containing RVD-encoding sequences and appropriate TALEN backbones [7,8] have made the TALEN technology widely available. Compatible backbones enable harnessing the additional specificity conferred by engineered heterodimeric FokI domains.…”

Epithelial DLD-1 Cells with Disrupted E-cadherin Gene Retain the Ability to Form Cell Junctions and Apico-basal Polarity

“…TALEN #1 and #2 plasmids were constructed by the protocol described by Cermak et al (2011) with modification (Sakuma et al, 2013a), and TALEN #3 plasmid was constructed as described by Sakuma et al (2013b) using the following kits obtained from Addgene (Cambridge, MA, USA): Golden Gate TALEN and TAL Effector Kit (#1000000024) with TALEN Construction Accessory Pack (#1000000030) for TALEN #1 and #2; Platinum Gate TALEN Kit (#1000000043) for TALEN #3. CRISPR/Cas9 plasmid was constructed using the pX330 vector (Addgene, #42230) according to the method described by Cong et al (2013) with some modifications.…”

Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases

“…Potential TALEN target sites in the locus were searched using the TALEN Targeter program (https:// tale-nt.cac.cornell.edu/node/add/talen) (Doyle et al 2012) with the following parameters: (1) spacer length of 14-17, (2) repeat array length of 16-18, and (3) upstream base of T only. TAL repeats were assembled by the Golden Gate assembly method (Cermak et al 2011) with slight modifications (Sakuma et al 2013). Each module containing an RVD was excised from each module plasmid (pNI, pHD, pNN, and pNG vectors) using BsaI-HF (New England Biolabs, Ipswich, MA), and then the fragment was purified using NucleoSpin Gel and PCR Clean-up kit (MACHEREY-NAGEL, Düren, Germany).…”