DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins

Woo, Je Wook; Kim, Jung-Eun; Kwon, Sanghoon; Corvalán, Claudia; Cho, Seung Woo; Kim, Hye‐Ran; Kim, Sang-Gyu; Choe, Sung Sik; Kim, Jin-Soo

doi:10.1038/nbt.3389

Cited by 1,034 publications

(751 citation statements)

References 23 publications

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“…In the end, reproductive success depends on multiple traits and we hypothesize that combining optimal variants of all these traits will be needed to significantly improve tolerance of reproduction to LTMH. Traits may be transferred and stacked through marker assisted breeding or by application of newly developed genetic modification methods (Sander and Joung 2014;Woo et al 2015).…”

Section: Resultsmentioning

confidence: 99%

Exploring the natural variation for reproductive thermotolerance in wild tomato species

et al. 2018

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Climate change has become a serious threat for crop productivity worldwide. The increased frequency of heat waves strongly affects reproductive success and thus yield for many crop species, implying that breeding for thermotolerant cultivars is critical for food security. Insight into the genetic architecture of reproductive heat tolerance contributes to our fundamental understanding of the stress sensitivity of this process and at the same time may have applied value. In the case of tomato (Solanum lycopersicum), germplasm screenings for thermotolerance have often used yield as the main measured trait. However, due to the complex nature of yield and the relatively narrow genetic variation present in the cultivated germplasm screened, there has been limited progress in understanding the genetic basis of reproductive heat tolerance. Extending the screening to wild accessions of related species that cover a range of climatic conditions might be an effective approach to find novel, more tolerant genetic resources. The purpose of this study was to provide insight into the sensitivity of individual reproductive key traits (i.e. the number of pollen per flower, pollen viability and style protrusion) to heat-wave like long-term mild heat (LTMH), and determine the extent to which genetic variation exists for these traits among wild tomato species. We found that these traits were highly variable among the screened accessions. Although no overall thermotolerant species were identified, several S. pimpinellifolium individuals outperformed the best performing cultivar in terms of pollen viability under LTMH. Furthermore, we reveal that there has been local adaptation of reproductive heat tolerance, as accessions from lower elevations and higher annual temperature are more likely to show high pollen viability under LTMH.

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Section: Resultsmentioning

confidence: 99%

Exploring the natural variation for reproductive thermotolerance in wild tomato species

et al. 2018

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“…However, the deregulation shown in the Argentine regulations would be likely if it is shown that the plasmid-derived DNA is not present in the final product. Moreover, the deregulation would be more likely if researchers use CRISPR/Cas9 in the form of a ribonucleoprotein which can be delivered into plant cells without the use of a DNA vector (Woo et al, 2015). We comprehensively surveyed the regulatory movements in relation to genome-edited crops or crop breeding by genome editing (Table 2).…”

Section: Future Global Regulatory Landscape Regarding Genome-edited Cmentioning

confidence: 99%

A future scenario of the global regulatory landscape regarding genome-edited crops

2016

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ABSTRACT. The global agricultural landscape regarding the commercial cultivation of genetically modified (GM) crops is mosaic. Meanwhile, a new plant breeding technique, genome editing is expected to make genetic engineering-mediated crop breeding more socially acceptable because it can be used to develop crop varieties without introducing transgenes, which have hampered the regulatory review and public acceptance of GM crops. The present study revealed that product-and process-based concepts have been implemented to regulate GM crops in 30 countries. Moreover, this study analyzed the regulatory responses to genome-edited crops in the USA, Argentina, Sweden and New Zealand. The findings suggested that countries will likely be divided in their policies on genome-edited crops: Some will deregulate transgene-free crops, while others will regulate all types of crops that have been modified by genome editing. These implications are discussed from the viewpoint of public acceptance.

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“…The CRISPR-Cas system can be stably or transiently transfected into the plant cells as nucleic acids (plasmids or DNA fragments) [6,73] or pre-assembled ribonucleoprotein complexes (RNPs) [74][75][76].…”

Section: Type Of Delivered Moleculesmentioning

confidence: 99%

“…Using Cas9 RNA or protein presents the advantage of not having to work on optimizing expression by defining sequences codon-optimized for the host plant and selecting appropriate promoters. Preassembled complexes of purified Cas9 protein and guide RNA (RNPs) were transfected into protoplasts of Arabidopsis thaliana, Nicotiana attenuata, Petunia x hybrida, grapevine, apple, lettuce and rice [74,76,95] and bombarded into immature embryos of bread and durum wheat [73,96], and maize [75]. In all cases, RNPs showed good cleavage efficiency (4-46%), with (when tested) no detectable mosaicism, suggesting a very early action of the nuclease before the first cell division [74,76].…”

Section: Transient Transformationmentioning

confidence: 99%

“…Mutagenesis frequency was lower (1.1%) than with DNA-based transient or stable transformation performed in the same conditions (about 3%), probably indicative of the instability of the RNAs in the plant cells [73]. Nevertheless, the rapid degradation of the RNAs is considered beneficial as it reduces the time during when the genome is exposed to nuclease activity, which limits the risk of generating off-target mutations [74]. So far, the only experiment reporting gene knock-in using a RNP complex was performed in maize by bombardment of immature embryos [75].…”

Section: Transient Transformationmentioning

confidence: 99%

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Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

Collonnier

Guyon‐Debast

Maclot

et al. 2017

Methods

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a b s t r a c tBeyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR-induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in.

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DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins

Cited by 1,034 publications

References 23 publications

Exploring the natural variation for reproductive thermotolerance in wild tomato species

Exploring the natural variation for reproductive thermotolerance in wild tomato species

A future scenario of the global regulatory landscape regarding genome-edited crops

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

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