Carbon nanotube–mediated DNA delivery without transgene integration in intact plants

Demirer, Gözde S.; Zhang, Huan; Goh, Natalie S.; González-Grandío, Eduardo; Landry, Markita P.

doi:10.1038/s41596-019-0208-9

Cited by 164 publications

(174 citation statements)

References 34 publications

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“…Obviously, an efficient regeneration protocol from calli into plants would eventually allow screening and validating genetically modified plants by advanced allele mining approaches previously used in TILLING projects (Nida et al, 2016). Moreover, new Cas9 delivery systems, which have recently been developed like nanomaterials delivery(Demirer et al, 2019) and Haploid-Inducer Mediated Genome Editing (IMGE)(Wang et al, 2019)(Kelliher et al, 2019) can be utilized to enhance Cas9 delivery system and allow better penetration. On top of that, HDR process is less frequent in somatic cells then NHEJ, therefore it is requiring a procedure that allows increasing HDR over NHEJ pathway e.g.…”

Section: Discussionmentioning

confidence: 99%

RECAS9: Recombining wild species introgression via mitotic gene editing in barley

Lazar

Prusty

Bishara

et al. 2020

Preprint

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Genetic loci underlying variation in traits with agronomic importance or genetic risk factors in human diseases have been identified by linkage analysis and genome-wide association studies.However, narrowing down the mapping to the individual causal genes and variations within these is much more challenging, and so is the ability to break linkage drag between beneficial and unfavourable loci in crop breeding. We developed RECAS9 as a transgene-free approach for precisely targeting recombination events by delivering CRISPR/Cas9 ribonucleotide protein (RNP) complex into heterozygous mitotic cells for the barley (Hordeum vulgare) Heat3.1 locus. A wild species (H. spontaneum) introgression in this region carries the agronomical unfavourable tough rachis phenotype (non-brittle) allele linked with a circadian clock accelerating QTL near GIGANTEA gene. We delivered RNP, which was targeted between two single nucleotide polymorphism (SNPs), to mitotic calli cells by particle bombardment. We estimated recombination events by next generation sequencing (NGS) and droplet digital PCR (ddPCR). While NGS analysis grieved from confounding effects of PCR recombination, ddPCR analysis allowed us to associate RNP treatment on heterozygous individuals with significant increase of homologous directed repair (HDR) between cultivated and wild alleles, with recombination rate ranging between zero to 57%. These results show for the first time in plants a directed and transgene free mitotic recombination driven by Cas9 RNP, and provide a starting point for precise breeding and fine scale mapping of beneficial alleles from crop wild relatives. Amunugama R, Fishel R (2012) Homologous recombination in eukaryotes. Prog. Mol. Biol. Transl. Sci. Elsevier B.V., pp 155-206 Barcelo P, Lazzeri PA (1995) Transformation of cereals by microprojectile bombardment of immature inflorescence and scutellum tissues. Methods Mol Biol. Bloom JS, Boocock J, Treusch S, Sadhu MJ, Day L, Oates-Barker H, Kruglyak L (2019) Rare variants contribute disproportionately to quantitative trait variation in yeast. Elife 8: 8-10 Brachi B, Faure N, Horton M, Flahauw E, Vazquez A, Nordborg M, Bergelson J, Cuguen J, Roux F (2010) Linkage and association mapping of Arabidopsis thaliana flowering time in nature. PLoS Genet 6: 40 Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kühn R (2015) Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. Edwards KD, Lynn JR, Gyula P, Nagy F, Millar AJ (2005) Natural allelic variation in the temperature-compensation mechanisms of the Arabidopsis thaliana circadian clock. Genetics 170: 387-400 Fernandes JB, Séguéla-Arnaud M, Larchevêque C, Lloyd AH, Mercier R (2018) Unleashing meiotic crossovers in hybrid plants. Proc Natl Acad Sci U S A 115: 2431-2436 Fernández-Calleja M, Casas AM, Pérez-Torres A, Gracia MP, Igartua E (2019) Rachis brittleness in a hybrid-parent barley (Hordeum vulgare) breeding germplasm with different combinations at the non-brittle...

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Section: Discussionmentioning

confidence: 99%

RECAS9: Recombining wild species introgression via mitotic gene editing in barley

Lazar

Prusty

Bishara

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…It was also achieved via an Agrobacterium infiltration, which requires significantly less time and technical skill than any transgenesis protocol. In the future we are excited to explore non-Agrobacterium based nucleic acid delivery methods, such as carbon nanotubes 34 , to expand these tools to species that cannot be easily infected by Agrobacterium. The capacity to avoid transgenesis also enables some common issues associated with the generation of stable lines to be side stepped.…”

Section: Discussionmentioning

confidence: 99%

VipariNama: RNA vectors to rapidly reprogram plant morphology and metabolism

Khakhar

Wang

Swanson

et al. 2020

Preprint

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Synthetic transcription factors have great promise as tools to explore biological processes. By allowing precise alterations in gene expression, they can help elucidate relationships between gene expression and plant morphology or metabolism. However, the years-long timescales, high cost, and technical skill associated with plant transformation have dramatically slowed their use. In this work, we developed a new platform technology called VipariNama (ViN) in which RNA vectors are used to rapidly deploy synthetic transcription factors and reprogram gene expression in planta. We demonstrate how ViN vectors can direct activation or repression of multiple genes, systemically and persistently over several weeks, and in multiple plant species. We also show how this transcriptional reprogramming can create predictable changes to metabolic and morphological phenotypes in the model plants Nicotiana benthamiana and Arabidopsis thaliana in a matter of weeks. Finally, we show how a model of gibberellin signaling can guide ViN vectorbased reprogramming to rapidly engineer plant size in both model species as well as the crop Solanum lycopersicum (tomato). In summary, using VipariNama accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

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“…[197][198][199][200][201] Recently, scientists have developed many other genome editing tools, such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas9 or Cpf1 system, [202][203][204][205][206][207] TALENS (Transcriptional Activator-Like Effector Nucleases), [208] chemical mutagenesis-mediated TILLING (Targeting Induced Local Lesions In Genomes), [193,209] ZFN (Zinc Finger Nuclease), etc. [208,210] Carbon nanotube is likely to improve transformation Small Methods 2020, 4,1900514 efficiency in Chlamydomonas, [211] but related new technologies are still being tested. [212] Genomic sequencing of many microalgae has been completed: Thalassiosira pseudonana, [213] Cyanidioschyzon merolae, [214] Ostreococcus tauri, [215] Chlamydomonas, [49,216,217] Ostreococcus lucimarinus, [218] Phaeodactylum tricornutum, [219] Micromonas pusilla, etc.…”

Section: Genetic Engineering To Improve Hydrogen Productionmentioning

confidence: 99%

Microalgal Hydrogen Production

et al. 2020

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Microalgae, as facultative aerobic organisms, convert solar energy to produce biohydrogen under anaerobic condition. Biohydrogen is a kind of ideal clean and renewable energy source with great commercial potential. Many endeavors have been focused on improving the biohydrogen yields by various means. Here, the research history of hydrogen production by microalgae (including cyanobacteria and green algae), the characteristics of nitrogenase and hydrogenase, the mechanisms of hydrogen production, the technological progress, and the application of enzymatic, genetic, and metabolic engineering methods to improve hydrogen production by microalgae are reviewed. In addition, the regulation of anaerobic metabolisms of Chlamydomonas reinhardtii after the disruption of key enzymes functioning in the fermentative pathways is discussed. Finally, the main challenges and obstacles facing the more efficient production and commercialization of hydrogen production from microalgae in the future are proposed.

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Carbon nanotube–mediated DNA delivery without transgene integration in intact plants

Cited by 164 publications

References 34 publications

RECAS9: Recombining wild species introgression via mitotic gene editing in barley

RECAS9: Recombining wild species introgression via mitotic gene editing in barley

VipariNama: RNA vectors to rapidly reprogram plant morphology and metabolism

Microalgal Hydrogen Production

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