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.

scholarly journals Customization and improvement of the LbCas12a-crRNA system for efficient gene targeting in plants

2021 ◽  
Author(s):  
Tien Vu ◽  
Duong Doan ◽  
Mil 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 customize 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 of LbCas12a gRNAs, the use of chemical treatments, and the establishment of favorable experimental conditions for further enhancement of plant HDR to enable efficient GT in tomato.

scholarly journals Improvement of the LbCas12a-crRNA System for Efficient Gene Targeting in Tomato

2021 ◽  
Vol 12 ◽  
Author(s):  
Tien Van Vu ◽  
Duong Thi Hai Doan ◽  
Mil Thi Tran ◽  
Yeon Woo Sung ◽  
Young Jong Song ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Low Temperatures ◽  
Plant Genome ◽  
Experimental Conditions ◽  
Chemical Treatments ◽  
Efficient Gene ◽  
Targeted Deep Sequencing ◽  
Small Chemical ◽  
Selection Of ◽  
Selection Markers

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 advance our GT system by inhibiting the cNHEJ pathway with small chemical molecules such as NU7441. Further optimization of the GT is also possible with the treatment of silver nitrate possibly via its pronounced actions in ethylene inhibition and polyamine production. Importantly, the GT efficiency is significantly enhanced with the use of a temperature-tolerant LbCas12a (ttLbCas12a) that is capable of performing target cleavage even at low temperatures. Targeted deep sequencing, as well as conventional methods, are used for the assessment of the editing efficiency at both cell and plant levels. 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.

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

Efficient gene targeting in a Candida guilliermondii non-homologous end-joining pathway-deficient strain

2013 ◽  
Vol 35 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Emilien Foureau ◽  
Vincent Courdavault ◽  
Luisa Fernanda Rojas ◽  
Christelle Dutilleul ◽  
Andrew J. Simkin ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Candida Guilliermondii ◽  
End Joining ◽  
Efficient Gene ◽  
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.

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.

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