Lorena's TAL Papers
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Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster

Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster | Lorena's TAL Papers | Scoop.it
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TALENs made with NK to specify G have less activity than their NN-containing counterparts: Targeting G with TAL Effectors: A Comparison of Activities of TALENs Constructed with NN and NK Repeat Var...

TALENs made with NK to specify G have less activity than their NN-containing counterparts: Targeting G with TAL Effectors: A Comparison of Activities of TALENs Constructed with NN and NK Repeat Var... | Lorena's TAL Papers | Scoop.it

Christian et al, (2012)

suggested Tom Schreiber, thanks!

 

The DNA binding domain of Transcription Activator-Like (TAL) effectors can easily be engineered to have new DNA sequence specificities. Consequently, engineered TAL effector proteins have become important reagents for manipulating genomes in vivo. DNA binding by TAL effectors is mediated by arrays of 34 amino acid repeats. In each repeat, one of two amino acids (repeat variable di-residues, RVDs) contacts a base in the DNA target. RVDs with specificity for C, T and A have been described; however, among RVDs that target G, the RVD NN also binds A, and NK is rare among naturally occurring TAL effectors. Here we show that TAL effector nucleases (TALENs) made with NK to specify G have less activity than their NN-containing counterparts: fourteen of fifteen TALEN pairs made with NN showed more activity in a yeast recombination assay than otherwise identical TALENs made with NK. Activity was assayed for three of these TALEN pairs in human cells, and the results paralleled the yeast data. The in vivo data is explained by in vitro measurements of binding affinity demonstrating that NK-containing TAL effectors have less affinity for targets with G than their NN-containing counterparts. On targets for which G was substituted with A, higher G-specificity was observed for NK-containing TALENs. TALENs with different N- and C-terminal truncations were also tested on targets that differed in the length of the spacer between the two TALEN binding sites. TALENs with C-termini of either 63 or 231 amino acids after the repeat array cleaved targets across a broad range of spacer lengths – from 14 to 33 bp. TALENs with only 18 aa after the repeat array, however, showed a clear optimum for spacers of 13 to 16 bp. The data presented here provide useful guidelines for increasing the specificity and activity of engineered TAL effector proteins.


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Bacterial Delivery of TALEN Proteins for Human Genome Editing

Bacterial Delivery of TALEN Proteins for Human Genome Editing | Lorena's TAL Papers | Scoop.it

"Transcription Activator-Like Effector Nucleases (TALENs) are a novel class of sequence-specific nucleases that have recently gained prominence for its ease of production and high efficiency in genome editing. A TALEN pair recognizes specific DNA sequences and introduce double-strand break in the target site, triggering non-homologous end joining and homologous recombination. Current methods of TALEN delivery involves introduction of foreign genetic materials, such as plasmid DNA or mRNA, through transfection. Here, we show an alternative way of TALEN delivery, bacterial type III secretion system (T3SS) mediated direct injection of the TALEN proteins into human cells. Bacterially injected TALEN was shown to efficiently target host cell nucleus where it persists for almost 12 hours. Using a pair of TALENs targetingvenus gene, such injected nuclear TALENs were shown functional in introducing DNA mutation in the target site. Interestingly, S-phase cells seem to show greater sensitivity to the TALEN mediated target gene modification. Accordingly, efficiency of such genome editing can easily be manipulated by the infection dose, number of repeated infections as well as enrichment of S phase cells. This work further extends the utility of T3SS in the delivery of functional proteins into mammalian cells to alter their characters for biomedical applications."

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A single plant resistance gene promoter engineered to recognize multiple TAL effectors from disparate pathogens

A single plant resistance gene promoter engineered to recognize multiple TAL effectors from disparate pathogens | Lorena's TAL Papers | Scoop.it

"we demonstrate that three functionally distinct" TAL binding "boxes targeted by separate TAL effectors retain their function and specificity when combined into one promoter. Given that many economically important" plant pathogenic pathogenic xanthomonads" deliver multiple TAL effectors, the engineering of Resistance genes capable of recognizing multiple TAL effectors provides a potential approach for engineering broad spectrum and durable disease resistance"


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Tools for TAL effector design and target prediction - Methods

(via T. Lahaye, thx)

Booher & Bogdanove 2014

TAL effectors are transcription factors injected into plant cells by pathogenic bacteria during infection. They find their specific DNA targets via a string of contiguous, structural repeats that individually recognize single nucleotides (with some degeneracy) by virtue of polymorphisms at residue 13. The number of repeats and sequence of the amino acids at position 13 determine the nucleotide sequence of the DNA target. Due to this modularity, TAL effectors are readily engineered and have been used alone or as molecular fusions for targeted gene activation, gene repression, chromatin modification, chromatin tagging, and most broadly, for genome editing as TAL effector nucleases (TALENs). Several moderate and high-throughput cloning methods are in place for assembling TAL effector-based genetic constructs. Targeting is complicated to an extent by a general requirement for thymine to precede the DNA target, a requirement of TALENs to bind paired opposing sites separated by a defined range of distances, differential contributions of different repeat types to overall affinity, and a polarity to mismatch tolerance. Several computational tools are available online to aid in design and the identification of candidate off-target binding sites, as well as assembly and implementation. These tools vary in their approaches, capabilities, and relative utility for different types of TAL effector applications. Accuracy of off-target prediction is not well characterized yet for any of the tools and will require a better understanding of the qualitative and quantitative variation in the nucleotide preferences of individual repeats.


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