A library of TAL effector nucleases spanning the human genome

Abstract

Transcription activator-like (TAL) effector nucleases (TALENs) can be readily engineered to bind specific genomic loci, enabling the introduction of precise genetic modifications such as gene knockouts and additions. Here we present a genome-scale collection of TALENs for efficient and scalable gene targeting in human cells. We chose target sites that did not have highly similar sequences elsewhere in the genome to avoid off-target mutations and assembled TALEN plasmids for 18,740 protein-coding genes using a high-throughput Golden-Gate cloning system. A pilot test involving 124 genes showed that all TALENs were active and disrupted their target genes at high frequencies, although two of these TALENs became active only after their target sites were partially demethylated using an inhibitor of DNA methyltransferase. We used our TALEN library to generate single- and double-gene-knockout cells in which NF-κB signaling pathways were disrupted. Compared with cells treated with short interfering RNAs, these cells showed unambiguous suppression of signal transduction.

Targeted gene knockout in chickens mediated by TALENs

“…TALEN Construction. To construct TALEN plasmids for DsRed, B4GALT1, and β-actin genes, TALEN expression plasmids were assembled via the one-step Golden Gate cloning system, as described previously (16). Chicken OV TALENs were assembled using methods described in Sanjana and colleagues (35).…”

“…Novel approaches have been recently designed to efficiently produce animals that are genetically altered in specific genomic sequences; zinc-finger nuclease (ZFN) (8)(9)(10) and transcription activator-like effector nuclease (TALEN) (11)(12)(13)(14)(15)(16) are representative next-generation platforms for customized genomic editing in transgenic animals, as well as in cultured cells in vitro. ZFNs and TALENs containing a DNA-binding domain and Fok I nuclease domain are currently crucial tools to dissect individual gene function in complicated biological systems with targeted genetic deletions and alterations.…”

Genome-Editing Technologies: Principles and Applications

“…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). In addition, adenoviral vectors have also proven particularly useful for mediating TALEN delivery to hard-to-transfect cell types (Holkers et al 2014;Maggio et al 2016).…”

A practical guide to induced pluripotent stem cell research using patient samples

“…64 Currently, TALEN plasmids targeting 18 740 protein-coding human genes have been assembled using a high-throughput GoldenGate cloning system. 65 Delivery of these TALENs can be achieved by injection of DNA or mRNA encoding TALENs or even the TALEN proteins directly. 62,66,67 CRISPR The CRISPR system is another effective genome-editing tool, which utilizes Cas9 nuclease to cleave DNA and chimeric guide RNA (gRNA) to target Cas9 to a specific region in the genome.…”