TAL effector science

(via Tom Schreiber. Thanks!)

Mashimo et al, 2013

TAL Effector Nucleases (TALENs) are versatile tools for targeted gene editing in various species. However, their efficiency is still insufficient, especially in mammalian embryos. Here, we showed that combined expression of Exonuclease 1 (Exo1) with engineered site-specific TALENs provided highly efficient disruption of the endogenous gene in rat fibroblast cells. A similar increased efficiency of up to ~30% with Exo1 was also observed in fertilized rat eggs, and in the production of knockout rats for the albino (Tyr) gene. These findings demonstrate TALENs with Exo1 is an easy and efficient method of generating gene knockouts using zygotes, which increases the range of gene targeting technologies available to various species.

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Chen et al, 2013

Zinc-finger nucleases (ZFNs) and TAL effector nucleases (TALENs) have been shown to induce targeted mutations, but they have not been extensively tested in any animal model. Here, we describe a large-scale comparison of ZFN and TALEN mutagenicity in zebrafish. Using deep sequencing, we found that TALENs are significantly more likely to be mutagenic and induce an average of 10-fold more mutations than ZFNs. We observed a strong correlation between somatic and germ-line mutagenicity, and identified germ line mutations using ZFNs whose somatic mutations rates are well below the commonly used threshold of 1%. Guidelines that have previously been proposed to predict optimal ZFN and TALEN target sites did not predict mutagenicity in vivo. However, we observed a significant negative correlation between TALEN mutagenicity and the number of CpG repeats in TALEN target sites, suggesting that target site methylation may explain the poor mutagenicity of some TALENs in vivo. The higher mutation rates and ability to target essentially any sequence make TALENs the superior technology for targeted mutagenesis in zebrafish, and likely other animal models.

Four rice genes and eight Brachypodium genes were targeted to generate knockout mutations. A protoplast transient assay was developed in both rice and Brachypodium. TALEN-encoding constructs were introduced into protoplasts using polyethylene glycol (PEG). Seven out of eleven TALENs showed mutagenesis frequencies ranging from
4% to 14%. The TALENs targeting the 4 rice and 8 Brachypodium genes were transformed into plants. The constructs were introduced into embryonic cells of rice or Brachypodium using Agrobacterium tumefaciens.

Two rice TALENs separated by 1322 bp in the OsBADH2
gene, were co-introduced into rice protoplasts. These two TALEN pairs were next co-delivered into rice calli by particle bombardment. Ninety-eight hygromycin-resistant calli were screened after 6 weeks of selection, and three types of modifications were identified — simple modifications (small deletions or insertions) at either target site, large deletions of the sequence between the
two target sites and inversion of the sequence between the two target sites.

These results support the idea that TALEN-induced genomic deletions were mediated via NHEJ. Targeted deletions would make possible the selective removal of gene clusters and enables scientists to delete intergenic regions, introns, regulatory elements and noncoding RNAs.
To our knowledge, this is the first study demonstrating highly efficient targeted
knockouts in multiple genes in the model monocot species Brachypodium. We anticipate TALEN technology will make targeted gene modification a routine practice not just for these model monocots but also for economically important crops, such as maize and wheat.

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Meckler et al, 2013

Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ∼ NN ≫ NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 103-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches ‘standard’ RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5′-T. Another surprising observation was that base mismatches at the 5′ end of the target site had more disruptive effects on affinity than those at the 3′ end, particularly in designed TALEs. These results provide evidence that TALE–DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.