Designer TALEs enable discovery of cell death-inducer genes.

Transcription activator-like effectors (TALEs) in plant-pathogenic Xanthomonas bacteria activate expression of plant genes and support infection or cause a resistance response. PthA4AT is a TALE with a particularly short DNA-binding domain harboring only 7.5 repeats which triggers cell death in Nicotiana benthamiana; however, the genetic basis for this remains unknown. To identify possible target genes of PthA4AT that mediate cell death in N. benthamiana, we exploited the modularity of TALEs to stepwise enhance their specificity and reduce potential target sites. Substitutions of individual repeats suggested that PthA4AT-dependent cell death is sequence specific. Stepwise addition of repeats to the C-terminal or N-terminal end of the repeat region narrowed the sequence requirements in promoters of target genes. Transcriptome profiling and in silico target prediction allowed the isolation of two cell death inducer genes, which encode a patatin-like protein and a bifunctional monodehydroascorbate reductase/carbonic anhydrase protein. These two proteins are not linked to known TALE-dependent resistance genes. Our results show that the aberrant expression of different endogenous plant genes can cause a cell death reaction, which supports the hypothesis that TALE-dependent executor resistance genes can originate from various plant processes. Our strategy further demonstrates the use of TALEs to scan genomes for genes triggering cell death and other relevant phenotypes.

Targeted C•G-to-T•A base editing with TALE-cytosine deaminases in plants.

BACKGROUND: TALE-derived DddA-based cytosine base editors (TALE-DdCBEs) can perform efficient base editing of mitochondria and chloroplast genomes. They use transcription activator-like effector (TALE) arrays as programmable DNA-binding domains and a split version of the double-strand DNA cytidine deaminase (DddA) to catalyze C•G-to-T•A editing. This technology has not been optimized for use in plant cells. RESULTS: To systematically investigate TALE-DdCBE architectures and editing rules, we established a ß-glucuronidase reporter for transient assays in Nicotiana benthamiana. We show that TALE-DdCBEs function with distinct spacer lengths between the DNA-binding sites of their two TALE parts. Compared to canonical DddA, TALE-DdCBEs containing evolved DddA variants (DddA6 or DddA11) showed a significant improvement in editing efficiency in Nicotiana benthamiana and rice. Moreover, TALE-DdCBEs containing DddA11 have broader sequence compatibility for non-TC target editing. We have successfully regenerated rice with C•G-to-T•A conversions in their chloroplast genome, as well as N. benthamiana with C•G-to-T•A editing in the nuclear genome using TALE-DdCBE. We also found that the spontaneous assembly of split DddA halves can cause undesired editing by TALE-DdCBEs in plants. CONCLUSIONS: Altogether, our results refined the targeting scope of TALE-DdCBEs and successfully applied them to target the chloroplast and nuclear genomes. Our study expands the base editing toolbox in plants and further defines parameters to optimize TALE-DdCBEs for high-fidelity crop improvement.

TAL effector-dependent Bax gene expression in transgenic rice confers disease resistance to Xanthomonas oryzae pv. oryzae.

The hypersensitive response (HR) is a form of programmed cell death of plant cells occurring in the local region surrounding pathogen infection site to prevent the spread of infection by pathogens. Bax, a mammalian pro-apoptotic member of Bcl-2 family, triggers HR-like cell death when expressed in plants. However, constitutive expression of the Bax gene negatively affects plant growth and development. The Xa10 gene in rice (Oryza sativa) is an executor resistance (R) gene that confers race-specific disease resistance to Xanthomonas oryzae pv. oryzae strains harboring TAL effector gene AvrXa10. In this study, the Xa10 promoter was used to regulate heterologous expression of the Bax gene from mouse (Mus musculus) in Nicotiana benthamiana and rice. Cell death was induced in N. benthamiana after co-infiltration with the PXa10:Bax:TXa10 gene and the PPR1:AvrXa10:TNos gene. Transgenic rice plants carrying the PXa10:Bax:TXa10 gene conferred specific disease resistance to Xa10-incompatible X. oryzae pv. oryzae strain PXO99A(pHM1AvrXa10), but not to the Xa10-compatible strain PXO99A(pHM1). The resistance specificity was confirmed by the AvrXa10-dependent induction of the PXa10:Bax:TXa10 gene in transgenic rice. Our results demonstrated that the inducible expression of the Bax gene in transgenic rice was achieved through the control of the executor R gene promoter and the heterologous expression of the pro-apoptosis regulator gene in rice conferred disease resistance to X. oryzae pv. oryzae.

Differential manifestation of ocular phenotypes in TALEN-mediated p19arf knockout FVB/N and C57BL/6J mouse lines.

BACKGROUND: p19arf, primarily known as a tumor suppressor, has also been reported to play an essential role in normal development of mouse eyes. Consistently, lack of p19arf has been associated with ocular defects, but the mixed background of the knockout (KO) mouse strain used raised a concern on the accuracy of the phenotypes observed in association with the targeted gene due to genetic heterogeneity. OBJECT: We carried out a study to investigate into the effect of genetic background on the manifestation of p19arf KO associated phenotypes. METHODS: We characterized the phenotypes of novel p19arf KO mouse lines generated in FVB/N and C57BL/6J using a transcription activator-like effector nuclease (TALEN) system in comparison to the reported phenotypes of three other p19arf-deficient mouse lines generated using homologous recombination. RESULTS: Ninety-five percent of FVB/N-p19arf KO mice showed ocular opacity from week 4 after birth which worsened rapidly until week 6, while such abnormality was absent in C57BL/6J-p19arf KO mice up to the age of 26 weeks. Histopathological analysis revealed retrolental masses and dysplasia in the retinal layer in FVB/N-p19arf KO mice from week 4. Besides these, both strains developed normally from birth to week 26 without increased tumorigenesis except for a subcutaneous tumor found in a C57BL/6J-p19arf KO mouse. CONCLUSION: Our findings demonstrated surprisingly variable manifestation of p19arf-linked phenotypes between FVB/N and C57BL/6J mice, and furthermore between our mouse lines and the established lines, indicating a critical impact of genetic background on functional study of genes using gene targeting strategies in mice.

Visualization of Genomic Loci in Living Cells with BiFC-TALE.

Tracking the dynamics of genomic loci is essential for understanding a variety of cellular processes. However, earlier methods have all suffered from a low signal-to-background ratio (SBR), mainly caused by the background fluorescence from diffuse full-length fluorescent proteins in the nucleus. We have developed a novel method (BiFC-TALE) for labeling and tracking genomic loci in live mammalian cells, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies. Since only the sequences-targeted BiFC fragments can be pulled together by TALE modules to recombine intact fluorescent proteins, the background fluorescence in the living nucleus can be largely reduced, which significantly improves SBR. Using telomere and centromere labeling as examples, this unit describes in detail the design and implementation of BiFC-TALE system. © 2018 by John Wiley & Sons, Inc.

Identification of a cell-penetrating peptide applicable to a protein-based transcription activator-like effector expression system for cell engineering.

Cellular reprogramming is a promising technology in regenerative medicine, but most studies have been performed by using expression vectors. For future clinical applications, it is necessary to establish a system in which cell engineering can be manipulated without any risk of damaging the genome. Here, we identified a cell-penetrating peptide composed of 10 amino acids (RIFIHFRIGC) with nuclear trafficking activity and found that it was significantly more potent than a Tat-derived peptide or polyarginine peptide (R11). We named the peptide "nuclear trafficking peptide" (NTP) and applied it to a protein-based artificial transcription factor (NTP-ATF), which was composed of a transcription activator-like effector and transcription domain (VP64). An NTP-ATF designed to the proximal promoter region of the microRNA-302/367 cluster efficiently induced endogenous RNA expression at an extremely low concentration (0.25 nM), and repetitive treatment of mouse embryonic fibroblasts with NTP-ATF generated induced pluripotent stem-like cells, which gave chimeric mice. Together with the observation that recombinant NTP-ATF protein did not induce any apparent cytotoxicity, we propose that NTP-ATF is a promising system for cellular reprogramming applicable to regenerative medicine.

Designed Transcriptional Regulation in Mammalian Cells Based on TALE- and CRISPR/dCas9.

Transcriptional regulation lies at the center of many cellular processes and is the result of cellular response to different external and internal signals. Control of transcription of selected genes enables an unprecedented access to shape the cellular response. While orthogonal transcription factors from bacteria, yeast, plants, or other cells have been used to introduce new cellular logic into mammalian cells, the discovery of designable modular DNA binding domains, such as Transcription Activator-Like Effectors (TALEs) and the CRISPR system, enable targeting of almost any selected DNA sequence. Fusion or conditional association of DNA targeting domain with transcriptional effector domains enables controlled regulation of almost any endogenous or ectopic gene. Moreover, the designed regulators can be linked into genetic circuits to implement complex responses, such as different types of Boolean functions and switches. In this chapter, we describe the protocols for achieving efficient transcriptional regulation with TALE- and CRISPR-based designed transcription factors in mammalian cells.

Multiple Chemical Inducible Tal Effectors for Genome Editing and Transcription Activation.

Inducible modulation is often required for precise investigations and manipulations of dynamic biological processes. Transcription activator-like effectors (TALEs) provide a powerful tool for targeted gene editing and transcriptional programming. We designed a series of chemical inducible systems by coupling TALEs with a mutated human estrogen receptor (ERT2), which renders them 4-hydroxyl-tamoxifen (4-OHT) inducible for access of the genome. Chemical inducible genome editing was achieved via fusing two tandem ERT2 domains to customized transcription activator-like effector nuclease (TALEN), which we termed "Hybrid Inducible Technology" (HIT-TALEN). Those for transcription activation were vigorously optimized using multiple construct designs. Most efficient drug induction for endogenous gene activation was accomplished with minimal background activity using an optimized inducible TALE based SunTag system (HIT-TALE-SunTag). The HIT-SunTag system is rapid, tunable, selective to 4-OHT over an endogenous ligand, and reversible in drug induced transcriptional activation. Versatile systems developed in this study can be easily applied for editing and transcriptional programming of potentially any genomic loci in a tight and effective chemical inducible fashion.

DNA residence time is a regulatory factor of transcription repression.

Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation.

Comparative analysis of chimeric ZFP-, TALE- and Cas9-piggyBac transposases for integration into a single locus in human cells.

Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells.

Genome engineering with TAL-effector nucleases and alternative modular nuclease technologies.

Over three years following the discovery of the TAL code, artificial TAL effector DNA binding domains have emerged as the premier platform for building site-specific DNA binding polypeptides for use in biological research. Here, we provide an overview of TAL effector and alternative modular DNA binding domain (mDBD) technologies, focusing on their use in established and emerging architectures for building site-specific endonucleases for genome engineering applications. We also discuss considerations for choosing TAL effector/mDBD or alternative nuclease technologies for genome engineering projects ranging from basic laboratory gene editing of cultured cell lines to therapeutics. Finally, we highlight how the rapid pace of development of mDBD-based, such as monomeric TALENs (I-TevI-TAL), and more recently RNA-guided nucleases (CRISPR-Cas9) has led to a transition in the field of genome engineering towards development of the next generation of technologies aimed at controlling events that occur after targeted DNA breaks are made.