Transcription Activator-like Effector Repeat Research Articles

Mitochondrial base editor induces substantial nuclear off-target mutations.

DddA-derived cytosine base editors (DdCBEs)-which are fusions of split DddA halves and transcription activator-like effector (TALE) array proteins from bacteria-enable targeted C•G-to-T•A conversions in mitochondrial DNA1. However, their genome-wide specificity is poorly understood. Here we show that the mitochondrial base editor induces extensive off-target editing in the nuclear genome. Genome-wide, unbiased analysis of its editome reveals hundreds of off-target sites that are TALE array sequence (TAS)-dependent or TAS-independent. TAS-dependent off-target sites in the nuclear DNA are often specified by only one of the two TALE repeats, challenging the principle that DdCBEs are guided by paired TALE proteins positioned in close proximity. TAS-independent off-target sites on nuclear DNA are frequently shared among DdCBEs with distinct TALE arrays. Notably, they co-localize strongly with binding sites for the transcription factor CTCF and are enriched in topologically associating domain boundaries. We engineered DdCBE to alleviate such off-target effects. Collectively, our results have implications for the use of DdCBEs in basic research and therapeutic applications, and suggest the need to thoroughly define and evaluate the off-target effects of base-editing tools.

High-efficiency plastome base editing in rice with TAL cytosine deaminase

High-efficiency plastome base editing in rice with TAL cytosine deaminase

PthAW1, a Transcription Activator-Like Effector of Xanthomonas citri subsp. citri, Promotes Host-Specific Immune Responses.

Citrus canker disease caused by Xanthomonas citri subsp. citri is one of the most destructive diseases in citrus. X. citri subsp. citri pathotypes display different host ranges. X. citri subsp. citri strain A (XccA) causes canker disease in most commercial citrus varieties, whereas strain AW (XccAW), which is genetically similar to XccA, infects only lime and alemow. Understanding the mechanism that determines the host range of pathogens is critical to investigating and utilizing host resistance. We hypothesized that XccAW would undergo mutations in genes that restrict its host range when artificially inoculated into incompatible citrus varieties. To test this hypothesis, we used an experimental evolution approach to identify phenotypic traits and genetic loci associated with the adaptation of XccAW to incompatible sweet orange. Repeated inoculation and reisolation cycles improved the ability of three independent XccAW strains to colonize sweet orange. Adapted XccAW strains displayed increased expression of type III secretion system and effector genes. Genome sequencing analysis indicated that two of the adapted strains harbored mutations in pthAW1, a transcription activator-like effector (TALE) gene, that corresponded to the removal of one or two repeats from the central DNA-binding repeat region. Introduction of the original but not the adapted pthAW1 variants into XccA abolished its ability to cause canker symptoms in sweet orange, Meyer lemon, and clementine but not in other XccAW-resistant citrus varieties. The original pthAW1, when expressed in XccA, induced ion leakage and the expression of pathogenesis-related genes but had no effect on CsLOB1 expression in sweet orange. Our study has identified a novel host-specific avirulence TALE and demonstrated active adaptive rearrangements of the TALE repeat array during host adaptation.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Consequences of adaptation of TAL effectors on host susceptibility to Xanthomonas.

Transcription activator-like effectors (TALEs) are virulence factors of Xanthomonas that induce the expression of host susceptibility (S) genes by specifically binding to effector binding elements (EBEs) in their promoter regions. The DNA binding specificity of TALEs is dictated by their tandem repeat regions, which are highly variable between different TALEs. Mutation of the EBEs of S genes is being utilized as a key strategy to generate resistant crops against TALE-dependent pathogens. However, TALE adaptations through rearrangement of their repeat regions is a potential obstacle for successful implementation of this strategy. We investigated the consequences of TALE adaptations in the citrus pathogen Xanthomonas citri subsp. citri (Xcc), in which PthA4 is the TALE required for pathogenicity, whereas CsLOB1 is the corresponding susceptibility gene, on host resistance. Seven TALEs, containing two-to-nine mismatching-repeats to the EBEPthA4 that were unable to induce CsLOB1 expression, were introduced into Xcc pthA4:Tn5 and adaptation was simulated by repeated inoculations into and isolations from sweet orange for a duration of 30 cycles. While initially all strains failed to promote disease, symptoms started to appear between 9-28 passages in four TALEs, which originally harbored two-to-five mismatches. Sequence analysis of adapted TALEs identified deletions and mutations within the TALE repeat regions which enhanced putative affinity to the CsLOB1 promoter. Sequence analyses suggest that TALEs adaptations result from recombinations between repeats of the TALEs. Reintroduction of these adapted TALEs into Xcc pthA4:Tn5 restored the ability to induce the expression of CsLOB1, promote disease symptoms and colonize host plants. TALEs harboring seven-to-nine mismatches were unable to adapt to overcome the incompatible interaction. Our study experimentally documented TALE adaptations to incompatible EBE and provided strategic guidance for generation of disease resistant crops against TALE-dependent pathogens.

Engineered TALE Repeats for Enhanced Imaging-Based Analysis of Cellular 5-Methylcytosine.

Transcription‐activator‐like effectors (TALEs) are repeat‐based, programmable DNA‐binding proteins that can be engineered to recognize sequences of canonical and epigenetically modified nucleobases. Fluorescent TALEs can be used for the imaging‐based analysis of cellular 5‐methylcytosine (5 mC) in repetitive DNA sequences. This is based on recording fluorescence ratios from cell co‐stains with two TALEs: an analytical TALE targeting the cytosine (C) position of interest through a C‐selective repeat that is blocked by 5 mC, and a control TALE targeting the position with a universal repeat that binds both C and 5 mC. To enhance this approach, we report herein the development of novel 5 mC‐selective repeats and their integration into TALEs that can replace universal TALEs in imaging‐based 5 mC analysis, resulting in a methylation‐dependent response of both TALEs. We screened a library of size‐reduced repeats and identified several 5 mC binders. Compared to the 5 mC‐binding repeat of natural TALEs and to the universal repeat, two repeats containing aromatic residues showed enhancement of 5 mC binding and selectivity in cellular transcription activation and electromobility shift assays, respectively. In co‐stains of cellular SATIII DNA with a corresponding C‐selective TALE, this selectivity results in a positive methylation response of the new TALE, offering perspectives for studying 5 mC functions in chromatin regulation by in situ imaging with increased dynamic range.

Design and Application of DNA Modification-Specific Transcription-Activator-Like Effectors.

Transcription-activator like effectors (TALEs) are DNA-binding proteins used for genome targeting. TALEs contain a central domain of concatenated repeats, of which each selectively recognizes one nucleobase at the DNA major groove. Based on this simple and predictable interaction with little context dependence, TALEs offer programmable targeting of user-defined DNA sequences. Since many epigenetic DNA modifications protrude into the DNA major groove, natural and engineered TALE repeats can provide "epigenetic" selectivity, making TALEs a flexible platform to design probes for the analysis of epigenetic DNA modifications. Here, we describe guidelines for the design of TALE proteins with selectivity for epigenetic cytosine 5-modifications, the validation of their interaction with modified DNA nucleobases, and their employment in affinity enrichment assays. These techniques enable quantification of epigenetic nucleobases in user-defined genomic DNA sequences with nucleotide and strand resolution.

Chimerization Enables Gene Synthesis and Lentiviral Delivery of Customizable TALE-Based Effectors.

Designer effectors based on the DNA binding domain (DBD) of Xanthomonas transcription activator-like effectors (TALEs) are powerful sequence-specific tools with an excellent reputation for their specificity in editing the genome, transcriptome, and more recently the epigenome in multiple cellular systems. However, the repetitive structure of the TALE arrays composing the DBD impedes their generation as gene synthesis product and prevents the delivery of TALE-based genes using lentiviral vectors (LVs), a widely used system for human gene therapy. To overcome these limitations, we aimed at chimerizing the DNA sequence encoding for the TALE-DBDs by introducing sufficient diversity to facilitate both their gene synthesis and enable their lentiviral delivery. To this end, we replaced three out of 17 Xanthomonas TALE repeats with TALE-like units from the bacterium Burkholderia rhizoxinica. This was combined with extensive codon variation and specific amino acid substitutions throughout the DBD in order to maximize intra- and inter-repeat sequence variability. We demonstrate that chimerized TALEs can be easily generated using conventional Golden Gate cloning strategy or gene synthesis. Moreover, chimerization enabled the delivery of TALE-based designer nucleases, transcriptome and epigenome editors using lentiviral vectors. When delivered as plasmid DNA, chimerized TALEs targeting the CCR5 and CXCR4 loci showed comparable activities in human cells. However, lentiviral delivery of TALE-based transcriptional activators was only successful in the chimerized form. Similarly, delivery of a chimerized CXCR4-specific epigenome editor resulted in rapid silencing of endogenous CXCR4 expression. In conclusion, extensive codon variation and chimerization of TALE-based DBDs enables both the simplified generation and the lentiviral delivery of designer TALEs, and therefore facilitates the clinical application of these tools to precisely edit the genome, transcriptome and epigenome.

Context and number of noncanonical repeat variable diresidues impede the design of TALE proteins with improved DNA targeting.

Transcription activator-like effector (TALE) proteins have been used extensively for targeted binding of fusion proteins to loci of interest in (epi)genome engineering. Such approaches typically utilize four canonical TALE repeat variable diresidue (RVD) types, corresponding to the identities of two key amino acids, to target each nucleotide. Alternate RVDs with improved specificity are desired. Here, we focused on seven noncanonical RVDs that have been suggested to have improved specificity for their target nucleotides. We used custom protein binding microarrays to characterize the DNA-binding activity of 65 TALEs containing these alternate or corresponding canonical RVDs at multiple positions to ~5,000 unique DNA sequences per protein. We found that none of the noncanonical thymine-targeting RVDs displayed stronger preference for thymine than did the canonical RVD. Of the noncanonical RVDs with putatively improved specificity for guanine, only EN and NH showed greater discrimination of guanine over adenine. This improved specificity, however, comes at a cost: more substitutions of a noncanonical RVD for a canonical RVD generally decreased the protein's DNA-binding activity. Our results highlight the need to investigate RVD-nucleotide specificities in multiple protein contexts and suggest that a balance between canonical and noncanonical RVDs is needed to build TALEs with improved specificity.

Programmable Protein-DNA Cross-Linking for the Direct Capture and Quantification of 5-Formylcytosine.

5-Formylcytosine (5fC) is an epigenetic nucleobase of mammalian genomes that occurs as intermediate of active DNA demethylation. 5fC uniquely interacts and reacts with key nuclear proteins, indicating functions in genome regulation. Transcription-activator-like effectors (TALEs) are repeat-based DNA binding proteins that can serve as probes for the direct, programmable recognition and analysis of epigenetic nucleobases. However, no TALE repeats for the selective recognition of 5fC are available, and the typically low genomic levels of 5fC represent a particular sensitivity challenge. We here advance TALE-based nucleobase targeting from recognition to covalent cross-linking. We report TALE repeats bearing the ketone-amino acid p-acetylphenylalanine (pAcF) that universally bind all mammalian cytosine nucleobases, but selectively form diaminooxy-linker-mediated dioxime cross-links to 5fC. We identify repeat-linker combinations enabling single CpG resolution, and demonstrate the direct quantification of 5fC levels in a human genome background by covalent enrichment. This strategy provides a new avenue to expand the application scope of programmable probes with selectivity beyond A, G, T and C for epigenetic studies.

Functional instability allows access to DNA in longer transcription Activator-Like effector (TALE) arrays.

Transcription activator-like effectors (TALEs) bind DNA through an array of tandem 34-residue repeats. How TALE repeat domains wrap around DNA, often extending more than 1.5 helical turns, without using external energy is not well understood. Here, we examine the kinetics of DNA binding of TALE arrays with varying numbers of identical repeats. Single molecule fluorescence analysis and deterministic modeling reveal conformational heterogeneity in both the free- and DNA-bound TALE arrays. Our findings, combined with previously identified partly folded states, indicate a TALE instability that is functionally important for DNA binding. For TALEs forming less than one superhelical turn around DNA, partly folded states inhibit DNA binding. In contrast, for TALEs forming more than one turn, partly folded states facilitate DNA binding, demonstrating a mode of 'functional instability' that facilitates macromolecular assembly. Increasing repeat number slows down interconversion between the various DNA-free and DNA-bound states.

Selective recognition of N4-methylcytosine in DNA by engineered transcription-activator-like effectors.

The epigenetic DNA nucleobases 5-methylcytosine (5mC) and N4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Complete, Programmable Decoding of Oxidized 5-Methylcytosine Nucleobases in DNA by Chemoselective Blockage of Universal Transcription-Activator-Like Effector Repeats.

5-Methylcytosine (5mC) and its oxidized derivatives are regulatory elements of mammalian genomes involved in development and disease. These nucleobases do not selectively modulate Watson-Crick pairing, preventing their programmable targeting and analysis by traditional hybridization probes. Transcription-activator-like effectors (TALEs) can be engineered for use as programmable probes with epigenetic nucleobase selectivity. However, only partial selectivities for oxidized 5mC have been achieved so far, preventing unambiguous target binding. We overcome this limitation by destroying and re-inducing nucleobase selectivity in TALEs via protein engineering and chemoselective nucleobase blocking. We engineer cavities in TALE repeats and identify a cavity that accommodates all eight human DNA nucleobases. We then introduce substituents with varying size, flexibility, and branching degree at each oxidized 5mC. Depending on the nucleobase, substituents with distinct properties effectively block TALE-binding and induce full nucleobase selectivity in the universal repeat. Successful transfer to affinity enrichment in a human genome background indicates that this approach enables the fully selective detection of each oxidized 5mC in complex DNA by programmable probes.

Evolution of Transcription Activator-Like Effectors in Xanthomonas oryzae.

Transcription activator-like effectors (TALEs) are secreted by plant–pathogenic Xanthomonas bacteria into plant cells where they act as transcriptional activators and, hence, are major drivers in reprogramming the plant for the benefit of the pathogen. TALEs possess a highly repetitive DNA-binding domain of typically 34 amino acid (AA) tandem repeats, where AA 12 and 13, termed repeat variable di-residue (RVD), determine target specificity. Different Xanthomonas strains possess different repertoires of TALEs. Here, we study the evolution of TALEs from the level of RVDs determining target specificity down to the level of DNA sequence with focus on rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) strains. We observe that codon pairs coding for individual RVDs are conserved to a similar degree as the flanking repeat sequence. We find strong indications that TALEs may evolve 1) by base substitutions in codon pairs coding for RVDs, 2) by recombination of N-terminal or C-terminal regions of existing TALEs, or 3) by deletion of individual TALE repeats, and we propose possible mechanisms. We find indications that the reassortment of TALE genes in clusters is mediated by an integron-like mechanism in Xoc. We finally study the effect of the presence/absence and evolutionary modifications of TALEs on transcriptional activation of putative target genes in rice, and find that even single RVD swaps may lead to considerable differences in activation. This correlation allowed a refined prediction of TALE targets, which is the crucial step to decipher their virulence activity.

Isolation of Human Genomic DNA Sequences with Expanded Nucleobase Selectivity.

We report the direct isolation of user-defined DNA sequences from the human genome with programmable selectivity for both canonical and epigenetic nucleobases. This is enabled by the use of engineered transcription-activator-like effectors (TALEs) as DNA major groove-binding probes in affinity enrichment. The approach provides the direct quantification of 5-methylcytosine (5mC) levels at single genomic nucleotide positions in a strand-specific manner. We demonstrate the simple, multiplexed typing of a variety of epigenetic cancer biomarker 5mC with custom TALE mixes. Compared to antibodies as the most widely used affinity probes for 5mC analysis, i.e., employed in the methylated DNA immunoprecipitation (MeDIP) protocol, TALEs provide superior sensitivity, resolution and technical ease. We engineer a range of size-reduced TALE repeats and establish full selectivity profiles for their binding to all five human cytosine nucleobases. These provide insights into their nucleobase recognition mechanisms and reveal the ability of TALEs to isolate genomic target sequences with selectivity for single 5-hydroxymethylcytosine and, in combination with sodium borohydride reduction, single 5-formylcytosine nucleobases.

Rapid and accurate synthesis of TALE genes from synthetic oligonucleotides.

Custom synthesis of transcription activator-like effector (TALE) genes has relied upon plasmid libraries of pre-fabricated TALE-repeat monomers or oligomers. Here we describe a novel synthesis method that directly incorporates annealed synthetic oligonucleotides into the TALE-repeat units. Our approach utilizes iterative sets of oligonucleotides and a translational frame check strategy to ensure the high efficiency and accuracy of TALE-gene synthesis. TALE arrays of more than 20 repeats can be constructed, and the majority of the synthesized constructs have perfect sequences. In addition, this novel oligonucleotide-based method can readily accommodate design changes to the TALE repeats. We demonstrated an increased gene targeting efficiency against a genomic site containing a potentially methylated cytosine by incorporating non-conventional repeat variable di-residue (RVD) sequences.

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats.

Improved specificity of TALE-based genome editing using an expanded RVD repertoire.

Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.

A Reason for Long Tales

Transcription Activator-Like Effectors (TALEs) are bacterial virulence factors containing a domain of repeats that recognize specific DNA sequences and reprogram transcription activation of invaded plant cells. TALE genes encode 5 to 30 repeats with average pairwise repeat identities greater than 91%. Most variability arises from only two positions termed Repeat Variable Diresidues (RVDs), which confer DNA binding specificity. Crystal structures of free and DNA-bound TALEs (Deng et al. Science 2012) show a large conformational change upon DNA binding. Thus, DNA binding is likely coupled to the free energy of tertiary structural change between the TALE repeats. We are interested in quantifying this relationship and relating it to folding cooperativity using nearest-neighbor (“Ising”) models.To investigate the length dependence of folding, we created a set of consensus TALE constructs of varying length. Solubilizing N- and C-terminal caps are needed to favor monomeric protein in solution as detected by sedimentation velocity analytical ultracentrifugation. Capped consensus TALE repeat constructs have alpha-helical secondary structure as measured by farUV CD. Urea-induced unfolding transitions of TALE repeat arrays were measured and show cooperative unfolding transitions as well as increases in stability with length. These data are well-fitted by an Ising model, which separates the contributions of intrinsic and interfacial free energies. TALE repeats have an unfavorable intrinsic folding free energy of 5.3 kcal/mol and a favorable interfacial free energy of −6.8 kcal/mol. Using these values, the length dependence of TALE stability can be modeled, and shows TALEs under 5 repeats to be unfolded. Using the Ising parameters, we find that partially folded states with a single repeat unfolded are energetically accessible. Population of these partially folded states may be important for DNA binding.

Achieving single-nucleotide resolution of 5-methylcytosine detection with TALEs.

We report engineered transcription-activator-like effectors (TALEs) as the first DNA-binding molecules that detect 5-methylcytosine (mC) at single-nucleotide resolution with fully programmable sequence selectivity. This is achieved by a design strategy such that a single cytosine (C) in a DNA sequence is selectively interrogated for its mC-modification level by targeting with a discriminatory TALE repeat; other Cs are ignored by targeting with universal-binding TALE repeats.

Rapid and Efficient Assembly of Transcription Activator-Like Effector Genes by USER Cloning

Transcription activator-like effectors (TALEs) that were related to bacteria immune system have lately been employed in a promising approach of precise gene targeting. Because of the repetitive characteristics of TALEs, existing TALE assembly methods are either very complicated, time-consuming, or too tricky to be handled in common labs. Here, we reported a rapid, efficient and easy method for TALE assembly. This method takes advantage of uracil-specific excision reagent (USER), an enzyme that can cleave DNA constructs and create long, unique single-strand DNA overhangs. Upon USER treatment, the overhangs on each individual TALE repeat unit can be rejoined hierarchically to form pentamers in a ligation-independent manner. Eventually, three pentamers are assembled into a full TALE construct by Golden Gate cloning. TALE nucleases (TALENs) generated with this method exhibit high genome-editing activity in human cells such as HEK293FT cells. Using this method, we have successfully synthesized three TALEN pairs targeting endogenous Tet1 locus, and proved that all can specifically target Tet1 gene, though in various degree. Comparing to other methods of TALEN assembly, this one is much less labor intensive and fairly faster, and positive clones can be obtained at high efficiency within only two days. We thus contribute to an easier approach for effective TALENs synthesis, which may highly facilitate the wide application of TALEN technology in genome editing, especially for human cells that require precise targeting.