CRISPR/Cas9 Targeted Mutagenesis for Functional Genetics in Maize

Hunter, Charles T.

doi:10.3390/plants10040723

Cited by 7 publications

(5 citation statements)

References 25 publications

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“…Two independent transgenic lines were created, one targeting fps1 alone and one targeting both fps2 and fps3 . The components for mutagenesis were delivered using the pRGEB32‐BAR vector (Hunter, 2021; available at http://addgene.com). This vector contains Cas9 from Streptococcus pyogenes driven by the rice ubiquitin promoter, a gRNA driven by the rice U3 snoRNA (Pol III) promoter, and BAR (glufosinate‐ammonium resistance) driven by the 35S promoter (Hunter, 2021).…”

Section: Methodsmentioning

confidence: 99%

“…The components for mutagenesis were delivered using the pRGEB32‐BAR vector (Hunter, 2021; available at http://addgene.com). This vector contains Cas9 from Streptococcus pyogenes driven by the rice ubiquitin promoter, a gRNA driven by the rice U3 snoRNA (Pol III) promoter, and BAR (glufosinate‐ammonium resistance) driven by the 35S promoter (Hunter, 2021). Cloning and vector preparation was conducted according to Xie et al.…”

Section: Methodsmentioning

confidence: 99%

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Dedicated farnesyl diphosphate synthases circumvent isoprenoid‐derived growth‐defense tradeoffs in Zea mays

et al. 2022

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Zea mays (maize) makes phytoalexins such as sesquiterpenoid zealexins, to combat invading pathogens. Zealexins are produced from farnesyl diphosphate in microgram per gram fresh weight quantities. As farnesyl diphosphate is also a precursor for many compounds essential for plant growth, the question arises as to how Z. mays produces high levels of zealexins without negatively affecting vital plant systems. To examine if specific pools of farnesyl diphosphate are made for zealexin synthesis we made CRISPR/Cas9 knockouts of each of the three farnesyl diphosphate synthases (FPS) in Z. mays and examined the resultant impacts on different farnesyl diphosphate-derived metabolites. We found that FPS3 (GRMZM2G098569) produced most of the farnesyl diphosphate for zealexins, while FPS1 (GRMZM2G168681) made most of the farnesyl diphosphate for the vital respiratory co-factor ubiquinone. Indeed, fps1 mutants had strong developmental phenotypes such as reduced stature and development of chlorosis. The replication and evolution of the fps gene family in Z. mays enabled it to produce dedicated FPSs for developmentally related ubiquinone production (FPS1) or defense-related zealexin production (FPS3). This partitioning of farnesyl diphosphate production between growth and defense could contribute to the ability of Z. mays to produce high levels of phytoalexins without negatively impacting its growth.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Dedicated farnesyl diphosphate synthases circumvent isoprenoid‐derived growth‐defense tradeoffs in Zea mays

et al. 2022

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“…For designing gRNA primers, various freely available web tools have been used [14]. Several reports advocate the use of the CHOPCHOP web tool for designing primers for gRNA [20][21][22]. So far, we have used the CHOPCHOP web tool for designing primers for gRNA.…”

Section: Resultsmentioning

confidence: 99%

Genome Editing Platforms in Rice (Oryza sativa L.): Basic methodology and troubleshooting

Kumari

Prasad

Dwivedi

2022

Preprint

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Nowadays, genome editing approaches are one of the most frequently used for studying the function of a new gene(s) and for the development of elite mutant lines with desired traits. The technology has to boost up the craze among the researchers for editing the crop genome. However, information regarding the constructions of CRISPR/Cas9 gene cassette to develop edited rice plants is scattered. In the present study, we have shown a systematic stepwise protocol for designing gRNA, cloning of gRNA in CRISPR/Cas9 binary vector, Agrobacterium-mediated transformation, screening and confirmation of edited plants along with troubleshooting at each step to accelerate the application of the CRISPR/Cas9 system for rice improvement. The CHOPCHOP web tool was used for designing primers for gRNA. In this study, we are mentioning a specific trait for gene editing because we are giving overall easy and efficient protocols for generating edited plants for any trait. Plants with the presence of CaMV35S promoter, OsU3 promoter, PAT gene, and Cas9 gene were treated as gene-edited plants whereas the absence of the desired band in plants was treated as wild type. The performance of genome editing technology in the laboratory depends upon the systematic steps to finally find the desirable edited plant, and this simplified method of CRISPR/Cas9 mediated gene editing will accelerate functional genomics studies in rice.

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“…Currently, Agrobacterium-mediated transformation is widely used to generate gene-edited plants, but this has major constraints due to few maize inbred lines amenable for transformation, low transformation efficiency and lower editing frequency (Ayar et al, 2013;Peterson et al, 2021;Hunter, 2021). Recently, several Agrobacterium-mediated transformation methods have been improved for maize genome-editing.…”

Section: Technology or System Improved Advantage Citationmentioning

confidence: 99%

CRISPR-Cas technology opens a new era for the creation of novel maize germplasms

Wang

Tang

et al. 2022

Front. Plant Sci.

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Maize (Zea mays) is one of the most important food crops in the world with the greatest global production, and contributes to satiating the demands for human food, animal feed, and biofuels. With population growth and deteriorating environment, efficient and innovative breeding strategies to develop maize varieties with high yield and stress resistance are urgently needed to augment global food security and sustainable agriculture. CRISPR-Cas-mediated genome-editing technology (clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated)) has emerged as an effective and powerful tool for plant science and crop improvement, and is likely to accelerate crop breeding in ways dissimilar to crossbreeding and transgenic technologies. In this review, we summarize the current applications and prospects of CRISPR-Cas technology in maize gene-function studies and the generation of new germplasm for increased yield, specialty corns, plant architecture, stress response, haploid induction, and male sterility. Optimization of gene editing and genetic transformation systems for maize is also briefly reviewed. Lastly, the challenges and new opportunities that arise with the use of the CRISPR-Cas technology for maize genetic improvement are discussed.

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CRISPR/Cas9 Targeted Mutagenesis for Functional Genetics in Maize

Cited by 7 publications

References 25 publications

Dedicated farnesyl diphosphate synthases circumvent isoprenoid‐derived growth‐defense tradeoffs in Zea mays

Dedicated farnesyl diphosphate synthases circumvent isoprenoid‐derived growth‐defense tradeoffs in Zea mays

Genome Editing Platforms in Rice (Oryza sativa L.): Basic methodology and troubleshooting

CRISPR-Cas technology opens a new era for the creation of novel maize germplasms

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