Welcome To Website IAS

Hot news
Achievement

Independence Award

- First Rank - Second Rank - Third Rank

Labour Award

- First Rank - Second Rank -Third Rank

National Award

 - Study on food stuff for animal(2005)

 - Study on rice breeding for export and domestic consumption(2005)

VIFOTEC Award

- Hybrid Maize by Single Cross V2002 (2003)

- Tomato Grafting to Manage Ralstonia Disease(2005)

- Cassava variety KM140(2010)

Centres
Website links
Vietnamese calendar
Library
Visitors summary
 Curently online :  59
 Total visitors :  7652038

T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds
Tuesday, 2023/08/29 | 06:33:34

Manuel Rhiel, Kerstin Geiger, Geoffroy Andrieux, Julia Rositzka, Melanie Boerries, Toni Cathomen and Tatjana I. Cornu

Frontiers in Genome Editing; PUBLISHED 20 February 2023; DOI 0.3389/fgeed.2023.1130736

ABSTRACT

Transcription activator-like effector nucleases (TALENs) are programmable nucleases that have entered the clinical stage. Each subunit of the dimer consists of a DNA-binding domain composed of an array of TALE repeats fused to the catalytically active portion of the FokI endonuclease. Upon DNA-binding of both TALEN arms in close proximity, the FokI domains dimerize and induce a staggered-end DNA double strand break. In this present study, we describe the implementation and validation of TALEN-specific CAST-Seq (T-CAST), a pipeline based on CAST-Seq that identifies TALEN-mediated off-target effects, nominates off-target sites with high fidelity, and predicts the TALEN pairing conformation leading to off-target cleavage. We validated T-CAST by assessing off-target effects of two promiscuous TALENs designed to target the CCR5 and TRAC loci. Expression of these TALENs caused high levels of translocations between the target sites and various off-target sites in primary T cells. Introduction of amino acid substitutions to the FokI domains, which render TALENs obligate-heterodimeric (OH-TALEN), mitigated the aforementioned off-target effects without loss of on-target activity. Our findings highlight the significance of T-CAST to assess off-target effects of TALEN designer nucleases and to evaluate mitigation strategies, and advocate the use of obligate-heterodimeric TALEN scaffolds for therapeutic genome editing.

 

See https://www.frontiersin.org/articles/10.3389/fgeed.2023.1130736/full

 

FIGURE 1: CAST-Seq analysis for CCR5-targeting TALEN. (A) NGS-based genotyping of CCR5 and CCR2. Indicated is the fraction of alleles bearing Indels at the CCR5 on-target site and the off-target site in CCR2 in untreated (UT) T cells, and T cells edited with TALEN. (B) Structural variations. Circos plot illustrates CAST-Seq results with enlargement of the chromosome three region encompassing CCR5 and CCR2 loci. Lines represent chromosomal rearrangements with the CCR5 target site: OMTs with >20 hits in red, NBSs with >40 hits in grey, ambiguous classification (OMT/HMT) in yellow. Red and blue layers represent the alignment and homology scores, respectively. (C) Alignment. Illustrated is the CAST-Seq alignment for ANKRD55 (OMT#1) to two right TALEN arms. Mismatched bases are highlighted in red. Positions of primers P#1/P#2 for NGS validation are indicated. (D) Genotyping of ANKRD55. NGS was performed with primers P#1/P#2. (E) Coverage plot of ANKRD55. Plot shows chromosomal position vs. number of reads, the putative TALEN binding sites (green and purple dashed lines), as well as the positions of primers P#1/P#2 and P#3/P#4. Red vertical lines show boundary of region. (F) Genotyping of OMT#1. NGS was performed with primers P#3/P#4. *** specifies p-value<0.001 (Fisher’s exact test, Bonferroni corrected p-values).

 

Back      Print      View: 185

[ Other News ]___________________________________________________
  • Auxin depletion from leaf primordia contributes to organ patterning
  • Phytochrome controls alternative splicing to mediate light responses in Arabidopsis
  • Maternal temperature history activates Flowering Locus T in fruits to control progeny dormancy according to time of year
  • Identification and evaluation of quantitative trait loci underlying resistance to multiple HG types of soybean cyst nematode in soybean PI 437655
  • Factor analytic mixed models for the provision of grower information from national crop variety testing programs
  • Exploring the areas of applicability of whole genome prediction methods for Asian rice (Oryza sativa L.)
  • Investigation of terpene diversification across multiple sequenced plant genomes
  • Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism
  • Effect of the ahas Transgene on Biological Nitrogen Fixation and Yield of Soybean
  • Arabidopsis ROCK1 transports UDP-GlcNAc/UDP-GalNAc and regulates ER protein quality control and cytokinin activity
  • A peripheral endocannabinoid mechanism contributes to glucocorticoid-mediated metabolic syndrome
  • High-Resolution Linkage Map and Chromosome-Scale Genome Assembly for Cassava (Manihot esculenta Crantz) from 10 Populations
  • Analysis of the Transcriptome of Banana Fruit during Ripening
  • Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1
  • Salt Tolerant Gene in Soybean Identified
  • Climate change decouples oceanic primary and export productivity and organic carbon burial
  • Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus
  • 5-Hydroxymethylcytosine Is Not Present in Appreciable Quantities in Arabidopsis DNA
  • Fine mapping of the qLOP2 and qPSR2 1 loci associated with chilling stress tolerance of wild rice seedlings
  • Natural diversity in daily rhythms of gene expression contributes to phenotypic variation

 

Designed & Powered by WEBSO CO.,LTD