TAL effector science

Nowak et al, 2021

Xanthomonads inject transcription-activator like effectors (TALEs) into plant cells, where TALEs bind to effector binding elements (EBEs) in plant promoters and transcriptionally activate downstream host susceptibility genes to promote disease.

Haq et al, 2021

Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight in rice, delivers transcription activator-like effector (TALE) proteins into host cells to activate susceptibility or resistance (R) genes that promote disease or immunity, respectively. Nonhost plants serve as potential reservoirs of R genes; consequently, nonhost R genes may trap TALEs to trigger an immune response. In this study, we screened 17 Xoo TALEs for their ability to induce a hypersensitive response (HR) in the nonhost plant Nicotiana benthamiana (Nb); only AvrXa10 elicited an HR when transiently expressed in Nb. The HR generated by AvrXa10 required both the central repeat region and the activation domain, suggesting a specific interaction between AvrXa10 and a potential R-like gene in nonhost plants. Evans blue staining and ion leakage measurements confirmed that the AvrXa10-triggered HR was a form of cell death, and the transient expression of AvrXa10 in Nb induced immune responses. Genes targeted by AvrXa10 in the Nb genome were identified by transcriptome profiling and prediction of effector binding sites. Using several approaches (in vivo reporter assays, electrophoretic mobility-shift assays, targeted designer TALEs, and on-spot gene silencing), we confirmed that AvrXa10 targets NbZnFP1, a C2H2-type zinc finger protein that resides in the nucleus. Functional analysis indicated that overexpression of NbZnFP1 and its rice orthologs triggered cell death in rice protoplasts. An NbZnFP1 ortholog was also identified in tomato and was specifically activated by AvrXa10. These results demonstrate that NbZnFP1 is a nonhost R gene that traps AvrXa10 to promote plant immunity in Nb.

(via T. Schreiber and T. Lahaye, thanks!)

Cheng et al. 2021

Transcription activator-like effectors (TALEs) have been effectively used for targeted genome editing, transcriptional regulation, epigenetic modification, and locus-specific DNA imaging. However, with the advent of the clustered regularly interspaced short palindromic repeat/Cas9 system, an easy-to-use tool with the same function as TALEs, TALEs have recently been abandoned because of their complexity, time consumption, and difficult handling in common labs. Here, we described a degenerated codon-based TALE assembly system for simple, rapid, and efficient TALE assembly. TALE trimers with nonrepetitive DNA sequences were amplified by PCR and sequentially assembled via Gibson assembly. Our method is cost-effective, requires only commonly used basic molecular biology reagents, and takes only 2 h from target sequence analysis to completion. This simple, rapid, and lab-friendly TALE assembly method will restore the value of TALEs in DNA targeting.

(via T. Schreiber, thx)

Tse et al, 2021

Architectural proteins alter the shape of DNA. Some distort the double helix by introducing sharp kinks. This can serve to relieve strain in tightly-bent DNA structures. Here, we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from Escherichia coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes.

(via T. Schreiber, thx)

Schwarze et al, 2021

Disruption of the C-C-Chemokine-receptor-5 (CCR5) gene induces resistance towards CCR5-tropic HIV. Here we optimised our previously described CCR5-Uco-TALEN and its delivery by mRNA electroporation. The novel variant, CCR5-Uco-hetTALEN features an obligatory heterodimeric Fok1-cleavage domain, which resulted in complete abrogation of off-target activity at previously found homodimeric as well as 7/8 in silico predicted, potential heterodimeric off-target sites, the only exception being highly homologous CCR2. Prevailing 18- and 10-bp deletions at the on-target site revealed microhomology-mediated end-joining as a major repair pathway. Notably, the CCR5Δ55–60 protein resulting from the 18-bp deletion was almost completely retained in the cytosol. Simultaneous cutting at CCR5 and CCR2 induced rearrangements, mainly 15-kb deletions between the cut sites, in up to 2% of T cells underlining the necessity to restrict TALEN expression. We optimised in vitro mRNA production and showed that CCR5-on- and CCR2 off-target activities of CCR5-Uco-hetTALEN were limited to the first 72 and 24–48 h post-mRNA electroporation, respectively. Using single-cell HRMCA, we discovered high rates of TALEN-induced biallelic gene editing of CCR5, which translated in large numbers of CCR5-negative cells resistant to HIVenv-pseudotyped lentiviral vectors. We conclude that CCR5-Uco-hetTALEN transfected by mRNA electroporation facilitates specific, high-efficiency CCR5 gene-editing (30%–56%) and it is highly suited for clinical translation subject to further characterisation of off-target effects.

Lee et al, 2021
DddA-derived cytosine base editors (DdCBEs), composed of the split interbacterial toxin DddAtox, transcription activator-like effector (TALE), and uracil glycosylase inhibitor (UGI), enable targeted C-to-T base conversions in mitochondrial DNA (mtDNA). Here, we demonstrate highly efficient mtDNA editing in mouse embryos using custom-designed DdCBEs. We target the mitochondrial gene, MT-ND5 (ND5), which encodes a subunit of NADH dehydrogenase that catalyzes NADH dehydration and electron transfer to ubiquinone, to obtain several mtDNA mutations, including m.G12918A associated with human mitochondrial diseases and m.C12336T that incorporates a premature stop codon, creating mitochondrial disease models in mice and demonstrating a potential for the treatment of mitochondrial disorders.

Wang et al., 2021

Transcription activator-like effector (TALE) nucleases (TALENs) efficiently recognize and cleave DNA in a sequence-dependent manner. However, current TALE custom synthesis methods are either complicated or expensive. Here we report a simple and low-cost method for TALE construct assembly. This method utilizes the denaturation/reannealing nature of double-stranded DNA to create a unique single-stranded DNA overhang for proper ordering of TALEmonomers in an engineered multimer.We successfully synthesized two TALEN pairs targeting the endogenous TET1
locus in human embryonic kidney cells and demonstrated their editing efficiency. Our method provides an alternative simple, low-cost method for effective TALEN assembly, which may improve the application of TALE-based technology.

(via T. Schreiber, thx)

Zhang et al, 2020

This study developed a new rapid transcription activator-like effector nuclease (TALEN) preparation protocol by thoroughly redesigning the widely used Golden Gate TALEN and TAL Effector Kit 2.0. The new protocol can be used to prepare any custom 18-bp binding TALENs in just one day (about 12 h), more rapidly than CRISPR. This protocol used a set of linear monomers, a final TALE-FokI backbone plasmid, and a pipeline to assemble the ready-to-use TALEN expression plasmid, which were all newly developed for this study. The set of linear monomers can be easily produced and reproduced by high-fidelity polymerase chain reaction (PCR) amplification in a 96-well plate using a pair of universal primers. Most important of all, our rapid TALEN construction pipeline can easily obtain many positive colonies with high efficiency (over 80%). By preparing five pairs of TALENs targeting five NF-κB genes (RELA, RELB, CREL,NFKB1, and NFKB2) and editing these genes in different cell lines (293T, HepG2, and PANC1), this study demonstrated that the new protocol has high efficiency, reproducibility, reliability, and applicability. Moreover, this study showed that the fabricated TALEN has much higher editing efficiency than CRISPR. Finally, this study developed a dual-tagging system for simultaneously tagging target proteins and successfully edited cells with a streptavidin-binding peptide (SBP) or AviTag via homology-directed repair, which could have wide applications in protein (antigen) preparation, immunoprecipitation, and a transcription factor chromatin immunoprecipitation assay.

An enzyme pulled from toxic bacteria can enter the organelle and perform single-nucleotide DNA swaps.

(via T. Schreiber, thx)

Wu et al, 2021

Transcription activator-like (TAL) effectors are major virulence factors secreted by the type III secretion systems of Xanthomonas oryzae pv. oryzicola (Xoc) and X. oryzae pv. oryzae (Xoo), causing bacterial leaf streak and bacterial blight, respectively, in rice. However, the knowledge of Xoc TAL effector function in promoting bacterial virulence remains limited.
Here, we isolated the highly virulent Xoc strain HGA4 from the outbreak region of Huanggang (Hubei, China), which contains four TAL effectors not found in the Chinese model strain RS105. Among these, Tal2b was selected for introduction into RS105, which resulted in a longer lesion length than that in the control.
Tal2b directly binds to the promoter region of the gene and activates the expression of OsF3H03g, which encodes 2-oxoglutarate-dependent dioxygenase in rice. OsF3H03g negatively regulates salicylic acid (SA)-related defense by directly reducing SA, and it plays a positive role in susceptibility to both Xoc and Xoo in rice.
OsF3H03g interacts with a uridine diphosphate-glycosyltransferase protein (OsUGT74H4), which positively regulates bacterial leaf streak susceptibility and may inactivate SA via glycosylation modification.