scholarly journals CRISPR/Cas9-mediated knockout of factors in non-homologous end joining pathway enhances gene targeting in silkworm cells

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Li Zhu ◽  
Hiroaki Mon ◽  
Jian Xu ◽  
Jae Man Lee ◽  
Takahiro Kusakabe
Keyword(s):  
Gene Targeting ◽  
End Joining ◽  
Non Homologous End Joining

scholarly journals Gene targeting facilitated by engineered sequence-specific nucleases and its potential for applications in crop improvement

2021 ◽  
Author(s):  
Daisuke Miki ◽  
Rui Wang ◽  
Jing Li ◽  
Dali Kong ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Agronomic Traits ◽  
Higher Plants ◽  
Crop Improvement ◽  
Strand Break ◽  
End Joining ◽  
Homology Directed Repair ◽  
Target Dna ◽  
Targeted Gene Editing ◽  
Non Homologous End Joining

Abstract Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world’s population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining (NHEJ), and are only rarely repaired via error-free homology-directed repair (HDR) if an appropriate donor template is provided. Gene targeting (GT), i.e., the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.

Efficient gene targeting in non-homologous end-joining-deficient Lipomyces starkeyi strains

2017 ◽  
Vol 63 (4) ◽  
pp. 751-763 ◽  
Author(s):  
Yoshifumi Oguro ◽  
Harutake Yamazaki ◽  
Satoshi Ara ◽  
Yosuke Shida ◽  
Wataru Ogasawara ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Lipomyces Starkeyi ◽  
End Joining ◽  
Efficient Gene ◽  
Non Homologous End Joining

Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for non-homologous end joining

2008 ◽  
Vol 45 (1) ◽  
pp. 68-75 ◽  
Author(s):  
François Villalba ◽  
Jérôme Collemare ◽  
Patricia Landraud ◽  
Karine Lambou ◽  
Viviane Brozek ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Magnaporthe Grisea ◽  
End Joining ◽  
Non Homologous End Joining

scholarly journals Homology-directed repair of a defective glabrous gene in Arabidopsis with Cas9-based gene targeting

2018 ◽  
Author(s):  
Florian Hahn ◽  
Marion Eisenhut ◽  
Otho Mantegazza ◽  
Andreas P.M. Weber
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Strand Break ◽  
Double Strand Break ◽  
In Planta ◽  
End Joining ◽  
Trichome Formation ◽  
Break Site ◽  
Repair Template ◽  
Non Homologous End Joining

ABSTRACTThe CRISPR/Cas9 system has emerged as a powerful tool for targeted genome editing in plants and beyond. Double-strand breaks induced by the Cas9 enzyme are repaired by the cell’s own repair machinery either by the non-homologous end joining pathway or by homologous recombination. While the first repair mechanism results in random mutations at the double-strand break site, homologous recombination uses the genetic information from a highly homologous repair template as blueprint for repair of the break. By offering an artificial repair template, this pathway can be exploited to introduce specific changes at a site of choice in the genome. However, frequencies of double-strand break repair by homologous recombination are very low. In this study, we compared two methods that have been reported to enhance frequencies of homologous recombination in plants. The first method boosts the repair template availability through the formation of viral replicons, the second method makes use of an in planta gene targeting approach. Additionally, we comparatively applied a nickase instead of a nuclease for target strand priming. To allow easy, visual detection of homologous recombination events, we aimed at restoring trichome formation in a glabrous Arabidopsis mutant by repairing a defective glabrous1 gene. Using this efficient visual marker, we were able to regenerate plants repaired by homologous recombination at frequencies of 0.12% using the in planta gene targeting approach, while both approaches using viral replicons did not yield any trichome-bearing plants.

scholarly journals Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting

2008 ◽  
Vol 36 (19) ◽  
pp. 6333-6342 ◽  
Author(s):  
Susumu Iiizumi ◽  
Aya Kurosawa ◽  
Sairei So ◽  
Yasuyuki Ishii ◽  
Yuichi Chikaraishi ◽  
...  
Keyword(s):  
Gene Targeting ◽  
End Joining ◽  
Dna Integration ◽  
Non Homologous End Joining

scholarly journals Agrobacterium tumefaciens T-DNA Integration and Gene Targeting in Arabidopsis thaliana Non-Homologous End-Joining Mutants

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Qi Jia ◽  
Paul Bundock ◽  
Paul J. J. Hooykaas ◽  
Sylvia de Pater
Keyword(s):  
Gene Targeting ◽  
Wild Type ◽  
End Joining ◽  
Dsb Repair ◽  
Critical Pathway ◽  
Dna Integration ◽  
Dna Insertion ◽  
Non Homologous End Joining ◽  
The Stability

In order to study the role of AtKu70 and AtKu80 in Agrobacterium-mediated transformation and gene targeting, plant lines with a T-DNA insertion in AtKu80 or AtKu70 genes were functionally characterized. Such plant lines lacked both subunits, indicating that heterodimer formation between AtKu70 and AtKu80 is needed for the stability of the proteins. Homozygous mutants were phenotypically indistinguishable from wild-type plants and were fertile. However, they were hypersensitive to the genotoxic agent bleomycin, resulting in more DSBs as quantified in comet assays. They had lower end-joining efficiency, suggesting that NHEJ is a critical pathway for DSB repair in plants. Both Atku mutants and a previously isolated Atmre11 mutant were impaired in Agrobacterium T-DNA integration via floral dip transformation, indicating that AtKu70, AtKu80, and AtMre11 play an important role in T-DNA integration in Arabidopsis. The frequency of gene targeting was not significantly increased in the Atku80 and Atku70 mutants, but it was increased at least 10-fold in the Atmre11 mutant compared with the wild type.

scholarly journals Improvement of the LbCas12a-crRNA system for efficient gene targeting in plants

2021 ◽  
Author(s):  
Tien Van Vu ◽  
Duong Thi Hai Doan ◽  
Mil Thi Tran ◽  
Yeon Woo Sung ◽  
Young Jong Song ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Plant Genome ◽  
End Joining ◽  
Experimental Conditions ◽  
Plant Gene ◽  
Chemical Treatments ◽  
Efficient Gene ◽  
Non Homologous End Joining ◽  
Selection Of ◽  
Selection Markers

Abstract Plant gene targeting (GT) can be utilized to precisely replace up to several kilobases of a plant genome. Recent studies using the powerful clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases significantly improved plant GT efficiency. However, GT for loci without associated selection markers is still inefficient. We previously utilized Lachnospiraceae bacterium Cas12a (LbCas12a) in combination with a replicon for tomato GT and obtained high GT efficiency with some selection markers. In this study, we optimize and advance our GT system by using a temperature-tolerant LbCas12a (ttLbCas12a) in combination with various crRNA forms and chemical treatments to suppress the canonical non-homologous end-joining pathway in tomato. Our work demonstrates the significance of the selection of gene scissors, the appropriate design and number of LbCas12a crRNAs, the use of chemical treatments, and the establishment of favorable experimental conditions for further enhancement of plant HDR to enable efficient GT in tomato.

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