TAL effector science

(via T. Schreiber, thx)

Wei et al, 2022

 Although ZFN and TALEN have been previously engineered to successfully cut and eliminate mtDNA in a programmable way5,6,7,8,9, correction of disease-causing point mutations in mtDNA remained challenging. Recently, DddA-derived cytosine base editors (DdCBEs) have been developed to specifically induce C-to-T conversion in mtDNA by fusion of sequence-programmable TALE and split deaminase derived from interbacterial toxins10.

In this study, we sought to test DdCBE’s ability for base editing of mtDNA in human embryos.

Taken together, our results demonstrate that DdCBE is an effective base editor for inducing point mutations in mtDNA of human embryos, and the efficiency is much higher in 8-cell embryos. Our 8-cell injection method could help generate mitochondrial disease models as well as derived embryonic stem cells for functional investigation of disease-associated mutations in mtDNA.

Liu, et al 2021
Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is that the 5′ nucleotide of the target is specific for thymine (T). TALE domains with alternative 5′ nucleotide specificities could expand the scope of DNA target sequences that can be bound by TALEs. Another drawback of TALEs is their tendency to bind and cleave off-target sequence, which hampers their clinical application and renders applications requiring high-fidelity binding unfeasible. This disclosure provides methods and strategies for the continuous evolution of proteins comprising DNA-binding domains, e.g., TALE domains. In some aspects, this disclosure provides methods and strategies for evolving such proteins under positive selection for a desired DNA-binding activity and/or under negative selection against one or more undesired (e.g., off-target) DNA-binding activities. Some aspects of this disclosure provide engineered TALE domains and TALEs comprising such engineered domains, e.g., TALE nucleases (TALENs), TALE transcriptional activators, TALE transcriptional repressors, and TALE epigenetic modification enzymes, with altered 5′ nucleotide specificities of target sequences. Engineered TALEs that target ATM with greater specificity are also provided.

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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.

Haq et al, 2021

Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight (BLB) in rice, delivers transcription activator-like effector (TALE) proteins into host cells to activate susceptibility (S) or resistance (R) genes for 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 Nicotiana benthamiana (Nb); only AvrXa10 elicited a HR when transiently expressed in Nb. The HR generated by AvrXa10 required both the central repeat region and activation domain, suggesting specific interaction of AvrXa10 with a potential R-like gene in nonhost plant. Evans blue staining and ion leakage measurements confirmed that the AvrXa10-triggered HR was a form of cell death, and transient expression of AvrXa10 in Nb induced immune responses. Based on transcriptome profiling and prediction of effector binding sites, genes targeted by AvrXa10 in the Nb genome were identified. 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 orthologues triggered cell death in rice protoplasts. A NbZnFP1 orthologue was also identified in tomato and was specifically activated by AvrXa10. The results demonstrate that NbZnFP1 is a nonhost R gene that traps AvrXa10 for plant immunity in Nb.

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Bhardwaj & Nain 2021

This review provides significant insights into the pros and cons of the two most popular genome editing tools TALENs and CRISPRs. This mini review suggests that, TALENs provides novel opportunities in the field of therapeutics being highly specific and sensitive toward DNA modifications. In this article, we will briefly explore the special features of TALENs that makes this tool indispensable in the field of synthetic biology. This mini review provides great perspective in providing true guidance to the researchers working in the field of trait improvement via genome editing.

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Erkes etl al 2021 (Preprint)

The yield of many crop plants can be substantially reduced by plant-pathogenic Xanthomonas bacteria. The infection strategy of many Xanthomonas strains is based on transcription activator-like effectors (TALEs), which are secreted into the host cells and act as transcriptional activators of plant genes that are beneficial for the bacteria. The modular DNA binding domain of TALEs contains tandem repeats, each comprising two hyper-variable amino acids. These repeat-variable diresidues (RVDs) bind to a continuous DNA stretch (a target box) and determine the specificity of a TALE. All available tools for the prediction of TALE targets within the host plant suffer from many false positives. In this paper we propose a strategy to improve prediction accuracy by considering the epigenetic state of the host plant genome in the region of the target box. To this end, we extend our previously published tool PrediTALE by two epigenetic features: (i) We allow for filtering target boxes according to chromatin accessibility and (ii) we allow for considering the methylation state of cytosines within the target box during prediction, since DNA methylation may affect the binding specificity of RVDs. Here, we determine the epigenetic features from publicly available DNase-seq, ATAC-seq, and WGBS-seq data in rice. We benchmark the utility of both epigenetic features separately and in combination, deriving ground-truth from RNA-seq infections studies in rice. We find an improvement for each individual epigenetic feature, but especially the combination of both. Having established an advantage in TALE target predicting considering epigenetic features, we use these data for promoterome and genome-wide scans by our new tool EpiTALE, leading to several novel putative virulence targets. Our results suggest that it would be worthwhile to collect condition-specific chromatin accessibility data and methylation information when studying putative virulence targets of Xanthomonas TALEs.

Becker & Boch, 2021
TALEN were the first easy-to-use genome editing technology and sparked the genome editing revolution. Their application in multiple species brought targeted mutagenesis to the attention of scientists worldwide. Key breakthrough successes of genome editing have since been achieved using TALEN, among these, the first commercialization of an edited crop and the first human cured from cancer. TALEN have since been largely replaced by the CRISPR technologies which are somewhat easier to build, much easier to multiplex, and have spawned multiple derived techniques. Nevertheless, the flexible and precise positioning of TALEN is unmatched, and thus they have continued to evolve to new functionalities. Here, we assemble essential facts, design guidelines as well as important past and exciting novel developments.

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Jain et al, 2021
Genome editing critically relies on selective recognition of target sites. However, despite recent progress, the underlying search mechanism of genome-editing proteins is not fully understood in the context of cellular chromatin environments. Here, we use single-molecule imaging in live cells to directly study the behavior of CRISPR/Cas9 and TALEN. Our single-molecule imaging of genome-editing proteins reveals that Cas9 is less efficient in heterochromatin than TALEN because Cas9 becomes encumbered by local searches on non-specific sites in these regions. We find up to a fivefold increase in editing efficiency for TALEN compared to Cas9 in heterochromatin regions. Overall, our results show that Cas9 and TALEN use a combination of 3-D and local searches to identify target sites, and the nanoscopic granularity of local search determines the editing outcomes of the genome-editing proteins. Taken together, our results suggest that TALEN is a more efficient gene-editing tool than Cas9 for applications in heterochromatin. While Cas9 outperforms TALENs in euchromatin, it is less efficient in heterochromatic regions. Here the authors, using single-molecule imaging, show that Cas9 uses a less efficient search strategy compared to TALENs in these regions.

Mahlzahn & Qi 2020

Transcription activator-like effector (TALE) is a DNA-binding domain that can be paired with a nuclease to create DNA double-strand breaks, or with an effector protein to alter gene transcription. The ability to precisely alter plant genomes and transcriptomes has provided many insights into gene function and has recently been utilized for crop improvement. Easy design and construction of TALE make the tool more accessible to a variety of researchers. Here, we describe two TALE-based systems: transcription activator-like effector nucleases (TALEN), for creating targeted mutations in a gene of interest, and multiplex TALE activation (mTALE-Act), for activating one or a few genes of interest at the transcription level. Assembly of these tools is based on Golden Gate cloning and Gateway recombination, which are cost-effective and streamlined cloning methods.