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Citations TALEN scout

Genome engineering via programmable nucleases has taken the life science community by storm. Transcription activator- like effector nucleases (TALENs) are designer nucleases that work in pairs, with each partner recognizing a DNA sequence 16-20 bp long and cleaving one of the DNA strands in the spacer region between the recognition sites. Te resulting double-strand breaks often lead to small insertions or deletions, disrupting the genes in which they occur. Now, a team from Seoul has created a plasmid library for producing TALENs with a high probability of efficacy for every gene in the human genome. Tis work, published by Kim et al. in Nature Biotechnology, began by optimizing TALEN archi- tecture to more precisely set the acceptable spacer length within the target site, which should increase specificity. Te authors then developed an algorithm to search for target sites in every human gene. Te search criteria were rigorous, requiring 40 bp target sequences with 12 or 13 bp spacers, at least 7 single-base mismatches with any off-target site, local- ization within the first 70% of the coding sequence and, for alternatively spliced genes, a target site within a common exon. With these restrictions, at least 1 target site could be found for 91% of human genes. Using modestly relaxed criteria, target sites for 18,740 genes were identified. Aſter preparing a set of 424 plasmids containing different TAL effector repeat domains, Kim and colleagues used PCR-free Golden Gate cloning to combine the appropriate sequence recognition modules with a nuclease domain to assemble 37,480 TALEN plasmids, representing TALEN pairs for all 18,740 genes. To test TALEN activity, the researchers transfected 104 TALEN pairs into HEK293 cells, finding 98.1% functional with an

Figure 2 Assembly of TALEN plasmids using a one-step Golden-Gate cloning system. (a) Scheme of Golden-Gate assembly of TALEN plasmids. A total of 424 TAL effector array plasmids (64 × 6 + 16 × 2 + 4 × 2) (KanR) and 8 FokI plasmids (AmpR) are used. (b) High- throughput Golden-Gate cloning in 96-well plates. Six TAL effector array plasmids and one FokI plasmid are mixed in each well. BsaI releases the TAL effector arrays and allows an ordered assembly of six TAL effector arrays into the FokI plasmid. (c) Pilot test of 15 TALENs using the T7E1 assay. Asterisks indicate the expected positions of DNA bands cleaved by T7E1. The numbers at the bottom of the gels indicate mutation frequencies measured by band intensities.

a b Disruptive technology Position 1 4x4x4 64 Position 2 Position 3 4x4x4 64 4x4x4 64

Position 4 Position 5 Position 6 4x4x4

4x4x4 4x4x4 64 64 4x4 16 4 64 4x4

16 4

One-step Golden-Gate cloning CTGA GCCA



NLS-HA tag

N term

Schematic of modular, Golden Gate-based cloning of TALEN plasmid library spanning the human genome. Source: Nature Biotechnology


Could switching the reagent used to disrupt cell membranes reset the standard protocol for proteomic analysis? Tat’s the contention explored by a group from the University of Ottawa in an article appearing in the Journal of Proteome Research. Traditionally, cells are lysed using a detergent such as SDS, which breaks open the cells, denatures the proteins, and solubilizes those that are hydrophobic. Because detergents tend to interfere with proteolytic enzymes, the surfactant is generally replaced by a chaotrope such as urea before trypsinization, keeping the proteins in solution without blocking prote- olysis. However, since mass spectrometry is disrupted by most chaotropes, these agents must also be removed. Prior efforts to simplify this workflow have not caught on since the proposed detergent/chaotrope replacements have their own limitations, such as incomplete solubilization and safety concerns. Now, Ning et al. propose using Amphiphols (APols), which are commer-

Vol. 54 | No. 6 | 2013

cells, we constructed 15 TALEN pairs, each targeting a different human gene. These TALENs each consisted of 18.5 RVD mod- ules and an obligatory heterodimeric FokI domain. We measured the genome-editing activities of these TALENs in HEK293 cells using T7 endonuclease I (T7E1), an enzyme that specifically recognizes and cleaves het- eroduplexes formed by the hybridization of wild-type and mutant DNA sequences18. HEK293 cells were trans- fected with plasmids that encoded each TALEN pair, and PCR prod- ucts amplified from genomic DNA were assayed by T7E1. Mutation frequencies were estimated from the intensities of cleaved bands relative to intact bands. Mutations were detected at all 15 sites at frequencies of 3.9–43% (Fig. 2c). This pilot experiment demonstrates that both the new TALEN architecture and the Golden-Gate assem- bly system are robust enough to allow genome-scale construction of TALENs.

Choice of TALEN target sites in the human genome To construct a human TALEN library—a genome-scale collection of human gene-targeting TALENs—we obtained the DNA sequences of 18,742 protein-coding genes from the databases compiled by the HUGO Gene Nomenclature Committee35 in March 2011 and at the National Center for Biotechnology Information36 in November 2011. We then developed a computational strategy to identify poten- tial target sites in each gene according to the following criteria (Supplementary Fig. 1). 1. To maximize gene-disrupting activities, we identified 40-bp

average mutation frequency of 16%. No off-target effects were detected. To demonstrate the potential of this resource, the authors created knockouts of genes associated with NK-κB signaling in cultured cells. In contrast to siRNA-treated cells, the TALEN-mutated cells showed complete suppression of NF-κB signal transduction. Researchers eager to create their own knockout clones should visit Tere, users need simply to search for a gene to pull up order information for the associated TALEN plasmid. Material transfer agreements and a fee to cover costs for the nonprofit entity providing the TALEN library resource apply.

1. Mix TAL effector array plasmids (KanR)

2. Add expression vectors and Golden-Gate reaction mixture

3. Assemble TALEN plasmid by thermocycling Indels (%) TALEN 4. Transformation 5. Overnight culture in LB (AmpR) TALEN 6. Mix glycerol and store at –80 °C Indels (%) 15 Indels (%) 9.5

* *


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Y. Kim et al. 2013. A library of TAL effector nucleases spanning the human genome. Nat Biotechnol. 31:251-8.

ACAT1 TALEN –+ – +

C H term



– + – +

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+ * 7.9 4. We chose target sites that resided in a common exon, in case

single gene is expressed in two or more splicing variants. These criteria were stringent enough to avoid poor sites that we

target sequences with 12- or 13-bp spacers in each gene, which are recognized by TALEN pairs that consist of two 18.5 RVDs, the largest TALEN monomers that can be assembled using our Golden-Gate cloning system. 2. Our algorithm searched for unique target sequences with

the minimum number of potential off-target sites. Thus, we avoided target sites with homologous sequences that can be found elsewhere in the genome. As a result, the most homologous potential off-target sites carried at least 7-base mismatches with the target site of choice. We defined potential off-target sites as any homodimeric or het- erodimeric half-sites separated by a 12- to 14-bp spacer. 3. To ensure complete gene disruption, we searched for sites located

upstream in each protein-coding region near the start codon and excluded sites that resided within the downstream 30% of each cod- ing sequence.


cially available polymers with some subunits functionalized with hydrophobic and hydro- philic groups: the former interact strongly with hydrophobic regions of proteins, the latter keep the complex soluble. To date, APols have been used in structural analysis of proteins, but Ning and colleagues hypoth- esized that the polymers, with a little help from sonication, might be capable of lysing cells and solubilizing proteins for trypsini- zation. Indeed, APols proved equivalent to RIPA buffer for lysing cultured cells, while only minimally affecting trypsin activity. Next, the authors compared sample prepa- ration with traditional methods versus the APol-based method. Te former comprised SDS extraction, acetone precipitation to remove the detergent, reconstitution in urea for trypsin digestion, desalting, and vacuum drying prior to mass spectrometry. By contrast, in the APol method, trypsin was added directly to the detergent- containing solution; aſter digestion, APols

not appropriate for site-specific gene knockout but were flexib enough to identify multiple sites in most genes. A genome-wide su vey identified at least one TALEN site that satisfied all of the criteria 17,120 out of 18,742 genes (91%). These sites were classified as group (Supplementary Tables 2,3 and Supplementary Method To identify additional target sites, we loosened the criteria and iden fied a total of 169,362 TALEN sites in 18,740 protein-coding gen (9.0 sites per gene on average). Our website (http://www.talenlibrar net/) reports up to 10 TALEN sites in each gene. The vast majori (98%) of these sites are targeted by 18.5/18.5 RVD TALENs. Of the sites 95% (160,712/169,362) do not have any homologous sites wi >85% sequence identity (that is, 6-base mismatches/≤40-bp sequenc in the human genome.

Genome-scale assembly of TALENs

Next we chose one target site per gene and assembled TALEN expre sion plasmids using the Golden-Gate cloning system. To facilita the process of large-scale assembly, we preferentially chose 18.5/18 RVD TALEN sites with 12-bp spacers in each gene. A total of 37,4 plasmids encoding 18,740 TALEN pairs were assembled in 96-w plates according to our optimized protocol (Fig. 2b an Supplementary Methods).

were removed by adding formic acid (at pH 3-4, APols aggregate and can be pelleted by centrifugation). Any concern that the accelerated protocol would compromise mass spectrometry was dismissed by data showing 30%-40% higher signal intensity and 40% more unique peptides in proteomic samples prepared using APols. Te authors attribute this difference to sample loss during the protein precipitation and desalting steps in the traditional protocol.

Z. Ning et al. 2013. From cells to peptides: “one-stop” integrated proteomic processing using amphipols. J Proteome Res. 12:1512-9.

tion with EcoRI restriction enzyme and (ii) DNA sequencing. W chose one E. coli transformant from each of the 399 96-well plate TALEN plasmids were purified from four colonies that were grow from each transformant, and then digested with EcoRI. Correct assembled TALEN plasmids yielded a diagnostic 2.5-kbp DNA ban Typically, at least two out of four plasmids isolated from each tran formant passed this test (Supplementary Fig. 2). To confirm t TAL effector array sequences in these plasmids, we subjected 2 plasmids that had passed the EcoRI test to dideoxy DNA sequencin

We performed quality control of the TALEN plasmids by (i) dige Ph.D.

Selected and wri ten by Nijsje Dorman,

BioTechniques 54: 297 (June 2013) doi 10.2144/000114035 * * – * * –


© 2013 Nature America, Inc. All rights reserved.

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