BurrH: a new modular DNA binding protein for genome engineering

Juillerat, Alexandre; Bertonati, Claudia; Dubois, Gwendoline; Guyot, Valérie; Thomas, Séverine; Valton, Julien; Beurdeley, Marine; Silva, George H.; Daboussi, Fayza; Duchâteau, Philippe

doi:10.1038/srep03831

Cited by 40 publications

(44 citation statements)

References 38 publications

(59 reference statements)

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“…This accords with the results of Juillerat et al. (22) using an in vivo reporter system. All further experiments were carried out using T 0 targets to allow optimal conditions for comparison to TALE controls.…”

Section: Resultsmentioning

confidence: 99%

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

Lange

Wolf

Dietze

et al. 2014

Nucleic Acids Research

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The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution.

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Section: Resultsmentioning

confidence: 99%

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

Lange

Wolf

Dietze

et al. 2014

Nucleic Acids Research

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“…Specifically, we generated VarSeTALEs repeat modules using sequences from Xanthomonas TALEs, Ralstonia TALE-likes (RipTALs (20)) and Burkholderia TALE-likes (BurrH/Bat1 and Bat2 (17,18)). Sequences of TALElikes repeats used as the raw material for design for the repeat arrays generated in this study are displayed in Supplementary Figure S1.…”

Section: Varsetale Repeat Arrays Contain Large Numbers Of Non-bsr Polmentioning

confidence: 99%

“…However, TALE-like proteins are produced by other bacteria, including members of the Ralstonia solanacearum species complex, whose TALE-likes show far greater non-BSR polymorphism than those of TALEs (11). TALE-likes are also found in Burkholderia rhizoxinica (17,18) and two unknown marine bacteria (19), and experimental evidence shows that their repeats can be embedded as functional elements into Xanthomonas TALEs. Non-BSR polymorphisms abound in TALE-like repeats at almost every position (Supplementary Figure S1), yet in all studied cases BSRs confer the same specificities in these TALE-like repeats as they do in Xanthomonas TALE repeats.…”

Section: Introductionmentioning

confidence: 99%

Exploiting the sequence diversity of TALE-like repeats to vary the strength of dTALE-promoter interactions

et al. 2017

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Designer transcription activator-like effectors (dTALEs) are programmable transcription factors used to regulate userdefined promoters. The TALE DNA-binding domain is a tandem series of amino acid repeats that each bind one DNA base. Each repeat is 33-35 amino acids long. A residue in the center of each repeat is responsible for defining DNA base specificity and is referred to as the base specificying residue (BSR). Other repeat residues are termed non-BSRs and can contribute to TALE DNA affinity in a non-base-specific manner. Previous dTALE engineering efforts have focused on BSRs. Non-BSRs have received less attention, perhaps because there is almost no non-BSR sequence diversity in natural TALEs. However, more sequence diverse, TALE-like proteins are found in diverse bacterial clades. Here, we show that natural non-BSR sequence diversity of TALEs and TALE-likes can be used to modify DNA-binding strength in a new form of dTALE repeat array that we term variable sequence TALEs (VarSeTALEs). We generated VarSeTALE repeat modules through random assembly of repeat sequences from different origins, while holding BSR composition, and thus base preference, constant. We used two different VarSeTALE design approaches combing either whole repeats from different TALE-like sources (inter-repeat VarSeTALEs) or repeat subunits corresponding to secondary structural elements (intra-repeat VarSeTALEs). VarSeTALE proteins were assayed in bacteria, plant protoplasts and leaf tissues. In each case, VarSeTALEs activated or repressed promoters with a range of activities. Our results indicate that natural non-BSR diversity can be used to diversify the binding strengths of dTALE repeat arrays while keeping target sequences constant.

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“…In this review, the major focus will be the use of CRISPR-Cas9, although other endonucleases are being used for specific applications. [5][6][7] From a screening perspective, the selection of the appropriate CRISPR/Cas technology (and the subsequent design and execution of the project) depends on the type of question being asked. Broadly speaking, most research groups 587916J BXXXX10.1177/1087057115587916Journal of Biomolecular ScreeningWade research-article2015 1 Screening Unit, Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia, Milan, Italy considering using genome editing in a screening context will have one of two different goals.…”

Section: Background and Introductionmentioning

confidence: 99%

High-Throughput Silencing Using the CRISPR-Cas9 System: A Review of the Benefits and Challenges

Wade

2015

SLAS Discovery

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The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has been seized upon with a fervor enjoyed previously by small interfering RNA (siRNA) and short hairpin RNA (shRNA) technologies and has enormous potential for high-throughput functional genomics studies. The decision to use this approach must be balanced with respect to adoption of existing platforms versus awaiting the development of more "mature" next-generation systems. Here, experience from siRNA and shRNA screening plays an important role, as issues such as targeting efficiency, pooling strategies, and off-target effects with those technologies are already framing debates in the CRISPR field. CRISPR/Cas can be exploited not only to knockout genes but also to up-or down-regulate gene transcription-in some cases in a multiplex fashion. This provides a powerful tool for studying the interaction among multiple signaling cascades in the same genetic background. Furthermore, the documented success of CRISPR/Cas-mediated gene correction (or the corollary, introduction of disease-specific mutations) provides proof of concept for the rapid generation of isogenic cell lines for highthroughput screening. In this review, the advantages and limitations of CRISPR/Cas are discussed and current and future applications are highlighted. It is envisaged that complementarities between CRISPR, siRNA, and shRNA will ensure that all three technologies remain critical to the success of future functional genomics projects.

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BurrH: a new modular DNA binding protein for genome engineering

Cited by 40 publications

References 38 publications

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

Exploiting the sequence diversity of TALE-like repeats to vary the strength of dTALE-promoter interactions

High-Throughput Silencing Using the CRISPR-Cas9 System: A Review of the Benefits and Challenges

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