Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy

Tochio, N.; Umehara, Kohei; Uewaki, Jun-ichi; Flechsig, Holger; Kondo, Masaharu; Dewa, Takehisa; Sakuma, Tetsushi; Yamamoto, Takashi; Saitoh, Takashi; Togashi, Yuichi; Tate, Shin‐ichi

doi:10.1038/srep37887

Cited by 9 publications

(10 citation statements)

References 30 publications

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“…For interrepeat VarSeTALEs the highly conserved leucine residue at position 29 ( Figure S1) was used as the breakpoint between repeats of different origins. Repeat subunits used in our intra-repeat VarSeTALEs correspond to secondary structural elements: short-helix (4-10), RVD loop (11)(12)(13)(14)(15), long-helix (16-28) and inter-repeat loop (29-1). Figure 1 illustrates the two design approaches using example sequences.…”

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

confidence: 99%

“…Indeed, out of our initial set of 13 intra-repeat VarSeTALEs, most were very poor repressors, not significantly different from the negative control dTALE (Figure 2; dotted line). However, a recent study has also demonstrated that non-RVD polymorphisms that disrupt inter-repeat interactions can increase the structural flexibility of a dTALE repeat array superhelix, enhancing DNA binding 14 .…”

Section: Comparing Varsetale Design Approaches: Intra Vs Inter-repeatmentioning

confidence: 99%

“…The power of non-RVDs to modify DNA binding parameters other than base-specificity has already been demonstrated in a few cases. A recent study showed that repeat polymorphisms at two non-RVD positions can increase the binding affinity between a dTALE repeat array and its target DNA 14 . Another study showed that extended TALE repeats, with additional non-RVD amino acids, can be used as a tool to provide optional target base skipping 15 .…”

Section: Introductionmentioning

confidence: 99%

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

Lange¹,

Schandry²,

Wunderlich³

et al. 2016

Preprint

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

confidence: 99%

Section: Comparing Varsetale Design Approaches: Intra Vs Inter-repeatmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Lange¹,

Schandry²,

Wunderlich³

et al. 2016

Preprint

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“…Alternatively, further study of nonBSRs could be used to build rational design rules, helping users to select promising combinations. For example, a recent study used a mix of in vitro binding assays and molecular dynamics simulations to understand the functional impact of certain non-BSR polymorphisms at two positions within dTALE arrays (15). The VarSeTALE set in this study have poor resolution over some areas, e.g.…”

Section: Future Improvements To Varsetale Designmentioning

confidence: 99%

“…A recent study showed that repeat polymorphisms at two non-BSR positions can increase the binding affinity between a dTALE repeat array and its target DNA (15). Another study showed that extended TALE repeats, with additional non-BSR amino acids, can be used as a tool to provide optional target base skipping (16).…”

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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Engineering Gene Therapy: Advances and Barriers

Shahryari

Burtscher

Nazari

et al. 2021

Advanced Therapeutics

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Currently, 33 gene‐therapy drugs/products have been approved in the clinic. Over 3000 completed and ongoing clinical gene therapy trials have been reported worldwide. The development/maturation of tools for gene manipulation and gene delivery, as well as molecular advances in the diagnosis of genetic diseases, have played a central role in gene therapy, which have greatly revolutionized the field. Versatile and diverse genetic tools for gene manipulations and deliveries, with the possibility of short and long‐term effects, are vital advantages of gene therapy tools, paving the road for the development of new therapies. However, efficacy and safety concerns, immune system responses, laborious approaches for developing and manufacturing, unknown gene‐therapy drug interactions with the host and the high cost of drugs/products, are significant barriers in gene therapy. Here, the authors review the attempts for engineering of the gene manipulations that have been undertaken in the last three decades and used in clinical trials focusing on 1) gene‐editing platforms, 2) viral gene delivery systems, and 3) nonviral gene tools. In this comprehensive review, the principles of these gene manipulation tools as well as advances and barriers for their application in modern therapies are discussed. Furthermore, trends and future directions in gene therapy are discussed.

show abstract

Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy

Cited by 9 publications

References 30 publications

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

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

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

Engineering Gene Therapy: Advances and Barriers

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