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

2020 ◽  
Vol 21 (3) ◽  
pp. 795 ◽  
Author(s):  
Yongxing Fang ◽  
Wladislaw Stroukov ◽  
Toni Cathomen ◽  
Claudio Mussolino
Keyword(s):  
Lentiviral Vectors ◽  
Repeat Sequence ◽  
Cellular Systems ◽  
Gene Synthesis ◽  
Synthesis Product ◽  
Transcription Activator ◽  
Specific Amino Acid ◽  
Repetitive Structure ◽  
Or Gene ◽  
Golden Gate Cloning

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.

A Decade Decoded: Spies and Hackers in the History of TAL Effectors Research

2019 ◽  
Vol 57 (1) ◽  
pp. 459-481 ◽  
Author(s):  
Alvaro L. Perez-Quintero ◽  
Boris Szurek
Keyword(s):  
Transcription Factors ◽  
Current Knowledge ◽  
Bacterial Colonization ◽  
Binding Mechanism ◽  
Transcription Activator ◽  
Plant Host ◽  
Tal Effectors ◽  
Specific Amino Acid ◽  
History Of ◽  
Weak Points

Transcription activator-like effectors (TALEs) from the genus Xanthomonas are proteins with the remarkable ability to directly bind the promoters of genes in the plant host to induce their expression, which often helps bacterial colonization. Metaphorically, TALEs act as spies that infiltrate the plant disguised as high-ranking civilians (transcription factors) to trick the plant into activating weak points that allow an invasion. Current knowledge of how TALEs operate allows researchers to predict their activity (counterespionage) and exploit their function, engineering them to do our bidding (a Manchurian agent). This has been possible thanks particularly to the discovery of their DNA binding mechanism, which obeys specific amino acid–DNA correspondences (the TALE code). Here, we review the history of how researchers discovered the way these proteins work and what has changed in the ten years since the discovery of the code. Recommended music for reading this review can be found in the Supplemental Material .

Homing endonucleases: DNA scissors on a mission

Genome ◽  
2012 ◽  
Vol 55 (8) ◽  
pp. 553-569 ◽  
Author(s):  
Mohamed Hafez ◽  
Georg Hausner
Keyword(s):  
Zinc Finger Nucleases ◽  
Homing Endonucleases ◽  
Gene Repair ◽  
Transcription Activator ◽  
Group I ◽  
Gene Knockouts ◽  
Or Gene ◽  
Triplex Forming Oligonucleotide ◽  
Self Splicing ◽  
Specific Sequences

Buried within the genomes of many microorganisms are genetic elements that encode rare-cutting homing endonucleases that assist in the mobility of the elements that encode them, such as the self-splicing group I and II introns and in some cases inteins. There are several different families of homing endonucleases and their ability to initiate and target specific sequences for lateral transfers makes them attractive reagents for gene targeting. Homing endonucleases have been applied in promoting DNA modification or genome editing such as gene repair or “gene knockouts”. This review examines the categories of homing endonucleases that have been described so far and their possible applications to biotechnology. Strategies to engineer homing endonucleases to alter target site specificities will also be addressed. Alternatives to homing endonucleases such as zinc finger nucleases, transcription activator-like effector nucleases, triplex forming oligonucleotide nucleases, and targetrons are also briefly discussed.

scholarly journals A 1·5 kb direct repeat sequence flanks the suppressor of forked gene at the euchromatin–heterochromatin boundary of the Drosophila melanogaster X chromosome

1996 ◽  
Vol 68 (3) ◽  
pp. 191-202 ◽  
Author(s):  
Mark Tudor ◽  
Andrew Mitchelson ◽  
Kevin O'hare
Keyword(s):  
Drosophila Melanogaster ◽  
X Chromosome ◽  
Polytene Chromosomes ◽  
Direct Repeat ◽  
Repeat Sequence ◽  
Metaphase Chromosomes ◽  
Homologous Sequences ◽  
Repeated Dna Sequence ◽  
Or Gene ◽  
Different Strains

SummaryA 1·5 kilobasepair repeated DNA sequence is duplicated in direct orientation so as to flank the suppressor of forked gene in the euchromatin–heterochromatin transition region on the X chromosome of Drosophila melanogaster. These two copies are almost identical, but DNA blotting, analysis of cloned sequences and database searches show that elsewhere in the genome, homologous sequences are poorly conserved. They are often associated with other repeats, suggesting that they may belong to a scrambled and clustered middle repetitive DNA family. The sequences do not appear to be related to transposable elements and their location in different strains is conserved. In situ hybridization to metaphase chromosomes shows that homologous sequences are concentrated in the pericentric regions of the autosomes and the X chromosome. The sequences are not significantly under-represented in DNA from polytene tissue and must lie in the replicated regions of polytene chromosomes. The almost perfect conservation of the two repeats around suppressor of forked in D. melanogaster suggests they arose by duplication or gene conversion. Suppression of recombination in this chromosomal region presumably allows this unusual organization to be stably maintained. In the X-ray induced allele, suppressor of forked-L26, the sequence between the repeats, including the gene, and one copy of the repeat have been deleted.

scholarly journals Chloroplast and mitochondrial DNA editing in plants

Nature Plants ◽  
2021 ◽  
Author(s):  
Beum-Chang Kang ◽  
Su-Ji Bae ◽  
Seonghyun Lee ◽  
Jeong Sun Lee ◽  
Annie Kim ◽  
...  
Keyword(s):  
Gene Editing ◽  
Unmet Need ◽  
Rrna Gene ◽  
Transcription Activator ◽  
Base Editing ◽  
Dna Editing ◽  
Point Mutagenesis ◽  
Golden Gate Cloning ◽  
Expression Plasmids ◽  
Plant Organelles

AbstractPlant organelles including mitochondria and chloroplasts contain their own genomes, which encode many genes essential for respiration and photosynthesis, respectively. Gene editing in plant organelles, an unmet need for plant genetics and biotechnology, has been hampered by the lack of appropriate tools for targeting DNA in these organelles. In this study, we developed a Golden Gate cloning system1, composed of 16 expression plasmids (8 for the delivery of the resulting protein to mitochondria and the other 8 for delivery to chloroplasts) and 424 transcription activator-like effector subarray plasmids, to assemble DddA-derived cytosine base editor (DdCBE)2 plasmids and used the resulting DdCBEs to efficiently promote point mutagenesis in mitochondria and chloroplasts. Our DdCBEs induced base editing in lettuce or rapeseed calli at frequencies of up to 25% (mitochondria) and 38% (chloroplasts). We also showed DNA-free base editing in chloroplasts by delivering DdCBE mRNA to lettuce protoplasts to avoid off-target mutations caused by DdCBE-encoding plasmids. Furthermore, we generated lettuce calli and plantlets with edit frequencies of up to 99%, which were resistant to streptomycin or spectinomycin, by introducing a point mutation in the chloroplast 16S rRNA gene.

scholarly journals Optical control of retrogradely infected neurons using drug-regulated “TLoop” lentiviral vectors

2014 ◽  
Vol 111 (10) ◽  
pp. 2150-2159 ◽  
Author(s):  
Ali Cetin ◽  
Edward M. Callaway
Keyword(s):  
Gene Expression ◽  
Viral Vectors ◽  
Lentiviral Vectors ◽  
Transduction Efficiency ◽  
Projection Neurons ◽  
Gene Products ◽  
Transcription Activator ◽  
Axon Terminals ◽  
The Face ◽  
Rabies Glycoprotein

Many approaches that use viral vectors to deliver transgenes have limited transduction efficiency yet require high levels of transgene expression. In particular, infection via axon terminals is relatively inefficient but is a powerful means of achieving infection of specific neuron types. Combining this with optogenetic approaches requires high gene expression levels that are not typically achieved with nontoxic retrogradely infecting vectors. We generated rabies glycoprotein-pseudotyped lentiviral vectors that use a positive feedback loop composed of a Tet promoter driving both its own tetracycline-dependent transcription activator (tTA) (“TLoop”) and channelrhodopsin-2-YFP (ChR2YFP). We show that TLoop vectors strongly express proteins in a drug-controllable manner in neurons that project to injection sites within the mouse brain. After initial infection, the virus travels retrogradely, stably integrates into the host genome, and expresses gene products. The expression is robust and allows optogenetic studies of neurons projecting to the location of virus injection, as demonstrated by fluorescence-targeted intracellular recordings. ChR2YFP expression did not cause observable signs of toxicity and continued for up to 6 mo after infection. Expression can be reversibly blocked by administration of doxycycline, if necessary, for expression of gene products that might be more toxic. Overall, we present a system that will allow researchers to achieve high levels of gene expression even in the face of inefficient viral transduction. The particular vectors that we demonstrate may enhance efforts to gain a precise understanding of the contributions of specific types of projection neurons to brain function.

scholarly journals Simple and efficient custom transcription activator-like effector gene synthesis via twin primer assembly

BioTechniques ◽  
2021 ◽  
Author(s):  
Song Wang ◽  
Yan Yu ◽  
Yuhualei Pan ◽  
Huan Wang ◽  
Yushang Zhao ◽  
...  
Keyword(s):  
Low Cost ◽  
Gene Synthesis ◽  
Synthesis Methods ◽  
Kidney Cells ◽  
Dependent Manner ◽  
Human Embryonic Kidney ◽  
Transcription Activator ◽  
Human Embryonic Kidney Cells ◽  
Editing Efficiency ◽  
Double Stranded Dna

Transcription activator-like effector (TALE) nucleases (TALENs) efficiently recognize and cleave DNA in a sequence-dependent manner. However, current TALE custom synthesis methods are either complicated or expensive. Here we report a simple and low-cost method for TALE construct assembly. This method utilizes the denaturation/reannealing nature of double-stranded DNA to create a unique single-stranded DNA overhang for proper ordering of TALE monomers in an engineered multimer. We successfully synthesized two TALEN pairs targeting the endogenous TET1 locus in human embryonic kidney cells and demonstrated their editing efficiency. Our method provides an alternative simple, low-cost method for effective TALEN assembly, which may improve the application of TALE-based technology.

scholarly journals Mobilization and Mechanism of Transcription of Integrated Self-Inactivating Lentiviral Vectors

2005 ◽  
Vol 79 (13) ◽  
pp. 8410-8421 ◽  
Author(s):  
Hideki Hanawa ◽  
Derek A. Persons ◽  
Arthur W. Nienhuis
Keyword(s):  
Lentiviral Vectors ◽  
Regulatory Elements ◽  
Trap Design ◽  
Promoter Sequences ◽  
Vector Integration ◽  
Vector Genome ◽  
Promoter Trap ◽  
A Cell ◽  
Or Gene ◽  
Cryptic Promoters

ABSTRACT Permanent genetic modification of replicating primitive hematopoietic cells by an integrated vector has many potential therapeutic applications. Both oncoretroviral and lentiviral vectors have a predilection for integration into transcriptionally active genes, creating the potential for promoter activation or gene disruption. The use of self-inactivating (SIN) vectors in which a deletion of the enhancer and promoter sequences from the 3′ long terminal repeat (LTR) is copied over into the 5′ LTR during vector integration is designed to improve safety by reducing the risk of mobilization of the vector genome and the influence of the LTR on nearby cellular promoters. Our results indicate that SIN vectors are mobilized in cells expressing lentiviral proteins, with the frequency of mobilization influenced by features of the vector design. The mechanism of transcription of integrated vector genomes was evaluated using a promoter trap design with a vector encoding tat but lacking an upstream promoter in a cell line in which drug resistance depended on tat expression. In six clones studied, all transcripts originated from cryptic promoters either upstream or within the vector genome. We estimate that approximately 1 in 3,000 integrated vector genomes is transcribed, leading to the inference that activation of cryptic promoters must depend on local features of chromatin structure and the constellation of nearby regulatory elements as well as the nature of the regulatory elements within the vector.

Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

2020 ◽  
Author(s):  
Nikolas Hundt
Keyword(s):  
Single Molecule ◽  
Cellular Systems ◽  
Single Molecule Imaging ◽  
Label Free ◽  
Measurement Sensitivity ◽  
Mass Distributions ◽  
Quantitative Measurements ◽  
Contrast Mechanism ◽  
Cellular Components ◽  
Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

Gene Myths or Gene Realities?

1994 ◽  
Vol 39 (9) ◽  
pp. 878-879 ◽  
Author(s):  
David C. Rowe
Keyword(s):  
Or Gene

Polymorphisms of the Plasminogen Activator Inhibitor- 1 Gene in Type 1 and Type 2 Diabetes, and in Patients with Diabetic Retinopathy

1994 ◽  
Vol 71 (06) ◽  
pp. 731-736 ◽  
Author(s):  
M W Mansfield ◽  
M H Stickland ◽  
A M Carter ◽  
P J Grant
Keyword(s):  
Diabetic Retinopathy ◽  
Plasminogen Activator Inhibitor ◽  
Allelic Frequency ◽  
Repeat Sequence ◽  
Dinucleotide Repeat ◽  
Plasminogen Activator Inhibitor 1 ◽  
The Difference ◽  
Pai 1

SummaryTo identify whether genotype contributes to the difference in PAI-1 levels in type 1 and type 2 diabetic subjects and whether genotype relates to the development of retinopathy, a Hind III restriction fragment length polymorphism and two dinucleotide repeat polymorphisms were studied. In 519 Caucasian diabetic subjects (192 type 1, 327 type 2) and 123 Caucasian control subjects there were no differences in the frequency of the Hind III restriction alleles (type 1 vs type 2 vs control: allele 1 0.397 vs 0.420 vs 0.448; allele 2 0.603 vs 0.580 vs 0.552) nor in the allelic frequency at either dinucleotide repeat sequence. In 86 subjects with no retinopathy at 15 years or more from diagnosis of diabetes and 190 subjects with diabetic retinopathy there was no difference in the frequency of Hind III restriction alleles (retinopathy present vs retinopathy absent: allele 1 0.400 vs 0.467; allele 2 0.600 vs 0.533) nor in the allelic frequencies at either dinucleotide repeat sequence. The results indicate that there is no or minimal influence of the PAI-1 gene on either PAI-1 levels or the development of diabetic retinopathy in patients with diabetes mellitus.

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