A simple cipher governs DNA recognition by TAL effectors
ABSTRACT TAL effectors of plant pathogenic bacteria in the genus Xanthomonas bind host DNA and activate genes that contribute to disease or turn on defense. Target specificity depends on an effector-variable number of typically 34 amino acid repeats, but the mechanism of recognition is not understood. We show that a repeat-variable pair of residues specifies the nucleotides in the target site, one pair to one nucleotide, with no apparent context dependence. Our finding represents a previously unknown mechanism for protein-DNA recognition that explains TAL effector specificity, enables target site prediction, and opens prospects for use of TAL effectors in research and biotechnology.
Full-textDOI: · Available from: Matthew J Moscou, Mar 11, 2015
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- "The effectors' binding domain consists of a variable number (13-28) of near-identical tandem repeats with each repeat comprising of 33 to 35 amino acids. The different types of these repeats feature a binding preference to one of the four nucleobases present in DNA (Boch et al., 2009; Moscou and Bogdanove, 2009). These specificities are defined by particular amino acids in positions 12 and 13, which thus have been coined as repeat variable diresidues (RVDs). "
ABSTRACT: Genome engineering is a breakthrough technology that facilitates the effective validation of functional DNA sequences and offers versatile possibilities for crop improvement through site-specific genetic modification. This emerging technology rests on synthetic endonucleases whose coding expression units can be customized to render these DNA cleavage enzymes specific for user-defined target sequences. The resultant cleavage site is then processed by the cells endogenous DNA repair machinery. The mechanisms of cellular DNA repair are either error-prone, which results in random sequence alterations at the genomic target site, or can be manipulated to facilitate the integration of a favored sequence. The novelty of genome engineering lies in its capacity to effect heritable genetic modifications at virtually any desired genomic position and, by repair template-mediated genome editing, to achieve the seamless correction, introduction, removal or exchange of any DNA sequence of choice. This review provides information on the currently available formats of synthetic endonucleases along with their working principles as well as some prospects they offer for plant research and crop improvement.Acta horticulturae 06/2015; 1087:71-81.
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- ", most of which contain 13e28 repeats and 33e35 amino acids per repeat ( Boch and Bonas , 2010 ) . Each repeat can recognize a single nucleotide in the target sequence , and the nucleotide spec - ificity is determined by a hypervariable region of two adjacent amino acids at positions 12 and 13 within each amino acid repeat , termed repeat - variable di - residue ( RVD ) ( Moscou and Bogdanove , 2009 ; Boch et al . , 2009 ; Morbitzer et al . "
ABSTRACT: Targeted genome editing mediated by clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) technology has emerged as one of the most powerful tools to study gene functions, and with potential to treat genetic disorders. Hearing loss is one of the most common sensory disorders, affecting approximately 1 in 500 newborns with no treatment. Mutations of inner ear genes contribute to the largest portion of genetic deafness. The simplicity and robustness of CRISPR/Cas9-directed genome editing in human cells and model organisms such as zebrafish, mice and primates make it a promising technology in hearing research. With CRISPR/Cas9 technology, functions of inner ear genes can be studied efficiently by the disruption of normal gene alleles through non-homologous-end-joining (NHEJ) mechanism. For genetic hearing loss, CRISPR/Cas9 has potential to repair gene mutations by homology-directed-repair (HDR) or to disrupt dominant mutations by NHEJ, which could restore hearing. Our recent work has shown CRISPR/Cas9-mediated genome editing can be efficiently performed in the mammalian inner ear in vivo. Thus, application of CRISPR/Cas9 in hearing research will open up new avenues for understanding the pathology of genetic hearing loss and provide new routes in the development of treatment to restore hearing. In this review, we describe major methodologies currently used for genome editing. We will highlight applications of these technologies in studies of genetic disorders and discuss issues pertaining to applications of CRISPR/Cas9 in auditory systems implicated in genetic hearing loss. Copyright © 2015. Published by Elsevier B.V.Hearing research 05/2015; 25. DOI:10.1016/j.heares.2015.04.016 · 2.85 Impact Factor
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- "bs_bs_banner (TALEs) are natural effector proteins secreted by numerous species of Xanthomonas, in order to modulate gene expression in host plants, and to facilitate bacterial colonization and survival (Boch and Bonas, 2010; Bogdanove et al., 2010). TALEs have revealed an elegant code linking the repetitive region of the TALEs with their target DNA-binding site (Boch et al., 2009; Moscou and Bogdanove, 2009). Two highly variable amino acids at positions 12 and 13, known as the repeat-variable di-residue (RVD) of mostly 33–35 amino acid tandem repeats, establish the base-recognition specificity of each unit. "
ABSTRACT: Bacillus nematocida B16 is able to dominate in the intestines of the worm Caenorhabditis elegans in 'Trojan horse' pathogenic mechanism. The adp is one candidate gene which potentially play a vital role in the colonization from our previous random mutagenesis screening results. To analyse the functional role of this gene, we constructed the adp knockout mutant through customized transcription activator-like effectors nucleases (TALEN), which has been successfully used in yeasts, nematodes, zebrafish and human pluripotent cells. Here, we first time report this knockout method in bacteria on this paper. Bioassay experiments demonstrated that the adp knockout mutant of B16 showed considerably lower colonization activity, reduced numbers of intestines and less than 80% nematocidal activity compared with the wild-type strain when infected for 48 h. However, no obvious change on proteolytic activity was observed in the mutant. Conversely, the complementation of adp gene restored most of the above deficient phenotypes. These results indicated that the adp gene was involved in surface adhesion and played a comparatively important role in colonizing host nematodes. Moreover, TALENs successfully disrupt target genes in bacteria. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.Microbial Biotechnology 05/2015; 8(4). DOI:10.1111/1751-7915.12282 · 3.21 Impact Factor