scholarly journals Molecular breeding of sporeless strains of Pleurotus ostreatus using a non-homologous DNA end-joining defective strain

2021 ◽  
Vol 20 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Fuga Yamasaki ◽  
Takehito Nakazawa ◽  
Masahiro Sakamoto ◽  
Yoichi Honda
Keyword(s):  
Gene Targeting ◽  
Pleurotus Ostreatus ◽  
Molecular Breeding ◽  
Single Gene ◽  
End Joining ◽  
Meiotic Progression ◽  
Targeted Gene Disruption ◽  
Efficient Gene ◽  
Efficient Breeding ◽  
Cultivated Mushrooms

AbstractGene targeting is useful to isolate strains with mutations in a gene of interest for efficient breeding. In this study, we generated msh4 or mer3 single-gene disruptant monokaryons using a Pleurotus ostreatus Δku80 strain for efficient gene targeting. Dikaryons of P. ostreatus Δmsh4×Δmsh4 or Δmer3×Δmer3 were isolated via backcrosses, and the number of basidiospores produced was measured. The number of basidiospores fell by an average 1/13.7 in the P. ostreatus Δmsh4×Δmsh4 dikaryons versus the P. ostreatus msh4+×Δmsh4 dikaryons, and 1/82.6 in the P. ostreatus Δmer3×Δmer3 dikaryons versus the P. ostreatus mer3+×Δmer3 dikaryons. To demonstrate the effects of ku80 disruption, P. ostreatus Δku80×Δku80 dikaryon strains were isolated and no significant effects on basidiospore production were observed. Fluorescence microscopy showed meiotic progression was arrested during prophase I in the msh4 or mer3 disruptants. To our knowledge, this is the first report on molecular breeding of sporeless strains in cultivated mushrooms using an efficient method for targeted gene disruption.

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

scholarly journals Efficient Gene Replacements in Toxoplasma gondii Strains Deficient for Nonhomologous End Joining

2009 ◽  
Vol 8 (4) ◽  
pp. 520-529 ◽  
Author(s):  
Barbara A. Fox ◽  
Jessica G. Ristuccia ◽  
Jason P. Gigley ◽  
David J. Bzik
Keyword(s):  
Dna Repair ◽  
Toxoplasma Gondii ◽  
Gene Targeting ◽  
Nonhomologous End Joining ◽  
Type I ◽  
Repair Pathway ◽  
Dna Breaks ◽  
End Joining ◽  
Γ Irradiation ◽  
Efficient Gene

ABSTRACT A high frequency of nonhomologous recombination has hampered gene targeting approaches in the model apicomplexan parasite Toxoplasma gondii. To address whether the nonhomologous end-joining (NHEJ) DNA repair pathway could be disrupted in this obligate intracellular parasite, putative KU proteins were identified and a predicted KU80 gene was deleted. The efficiency of gene targeting via double-crossover homologous recombination at several genetic loci was found to be greater than 97% of the total transformants in KU80 knockouts. Gene replacement efficiency was markedly increased (300- to 400-fold) in KU80 knockouts compared to wild-type strains. Target DNA flanks of only ∼500 bp were found to be sufficient for efficient gene replacements in KU80 knockouts. KU80 knockouts stably retained a normal growth rate in vitro and the high virulence phenotype of type I strains but exhibited an increased sensitivity to double-strand DNA breaks induced by treatment with phleomycin or γ-irradiation. Collectively, these results revealed that a significant KU-dependent NHEJ DNA repair pathway is present in Toxoplasma gondii. Integration essentially occurs only at the homologous targeted sites in the KU80 knockout background, making this genetic background an efficient host for gene targeting to speed postgenome functional analysis and genetic dissection of parasite biology.

scholarly journals Efficient genome editing with CRISPR/Cas9 in Pleurotus ostreatus

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tatpong Boontawon ◽  
Takehito Nakazawa ◽  
Chikako Inoue ◽  
Keishi Osakabe ◽  
Moriyuki Kawauchi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Pleurotus Ostreatus ◽  
Molecular Breeding ◽  
Heterologous Gene Expression ◽  
Homology Directed Repair ◽  
Guide Rnas ◽  
Efficient Gene ◽  
Fungal Protoplasts ◽  
Resistant Strains ◽  
Drug Resistant Strains

AbstractPleurotus ostreatus is one of the most commercially produced edible mushrooms worldwide. Improved cultivated strains with more useful traits have been obtained using classical breeding, which is laborious and time-consuming. Here, we attempted efficient gene mutagenesis using plasmid-based CRISPR/Cas9 as the first step for non-genetically modified (non-GM) P. ostreatus generation. Plasmids harboring expression cassettes of Cas9 and different single guide RNAs targeting fcy1 and pyrG were individually transferred into fungal protoplasts of the PC9 strain, which generated some strains exhibiting resistance to 5-fluorocytosine and 5-fluoroorotic acid, respectively. Genomic PCR followed by sequencing revealed small insertions/deletions or insertion of a fragment from the plasmid at the target site in some of the drug-resistant strains. The results demonstrated efficient CRISPR/Cas9-assisted genome editing in P. ostreatus, which could contribute to the molecular breeding of non-GM cultivated strains in the future. Furthermore, a mutation in fcy1 via homology-directed repair using this CRISPR/Cas9 system was also efficiently introduced, which could be applied not only for precise gene disruption, but also for insertions leading to heterologous gene expression in this fungus.

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.

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.

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