The present and potential future methods for delivering CRISPR/Cas9 components in plants

Sandhya, Dulam; Jogam, Phanikanth; Allini, Venkateswar Rao; Abbagani, Sadanandam; Alok, Anshu

doi:10.1186/s43141-020-00036-8

Cited by 101 publications

(53 citation statements)

References 94 publications

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“…In the present report, we show a positive correlation between the time of exposure to RNPs and the efficiency of site-directed mutagenesis in maize, as ascertained with Sanger sequencing. In previous reports referring to the use of RNPs in plant protoplasts ( Table 3 ), the authors used one or more different concentrations of RNPs and Cas9:gRNA ratios, but the effect of exposure time on mutation frequencies was not tested [ 29 , 30 ]. CRISPR RNPs were delivered to apple, grapevine, brassica sp., lettuce, tobacco, and rice plants at less or equal than 20 min exposure time and their efficiencies ranged between 0.1 and 40% [ 12 , 13 , 31 ].…”

Section: Discussionmentioning

confidence: 99%

PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts

Sant’Ana

Caprestano

Nodari

et al. 2020

Genes

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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated ones, especially plant cells, is still challenging. In a protoplast system, previous studies claimed that such pitfalls would be averted by delivering pre-assembled ribonucleoprotein complexes (RNPs) composed of purified recombinant Cas9 enzyme and in vitro transcribed guide RNA (gRNA) molecules. We, therefore, aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with polyethylene glycol (PEG) 4000. We performed an effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene, and by applying two different exposure times to RNPs. Using a low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between the exposure time and mutation frequency. The mutation frequency was gRNA sequence- and exposure time-dependent. In the present study, we demonstrated that the suitability of RNP transfection was proven as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for the selection of gRNA suitable for the editing of the gene of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively. Nevertheless, the large amplitude of mutations at the target site require scrutiny when checking mutations at off-target sites and potential safety concerns.

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Section: Discussionmentioning

confidence: 99%

PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts

Sant’Ana

Caprestano

Nodari

et al. 2020

Genes

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“…ODM has been successfully applied to several crops like maize (Zhu et al., 2000), rice (Okuzaki and Toriyama, 2004) and oilseed rape (Gocal et al., 2015). Multiple delivery systems for SDN components have been tested for different plant tissues, including PEG‐fusion, electroporation and biolistics (for a review of CRISPR/Cas9 component delivery methods see (Sandhya et al., 2020)).…”

Section: Introductionmentioning

confidence: 99%

Applicability of the EFSA Opinion on site‐directed nucleases type 3 for the safety assessment of plants developed using site‐directed nucleases type 1 and 2 and oligonucleotide‐directed mutagenesis

Naegeli¹,

Bresson²,

Dalmay³

et al. 2020

EFS2

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The European Commission requested the EFSA Panel on Genetically Modified Organisms (GMO) to assess whether section 4 (hazard identification) and the conclusions of EFSA's Scientific opinion on the risk assessment of plants developed using zinc finger nuclease type 3 technique (ZFN-3) and other site-directed nucleases (SDN) with similar function are valid for plants developed via SDN-1, SDN-2 and oligonucleotide-directed mutagenesis (ODM). In delivering this Opinion, the GMO Panel compared the hazards associated with plants produced via SDN-1, SDN-2 and ODM with those associated with plants obtained via both SDN-3 and conventional breeding. Unlike for SDN-3 methods, the application of SDN-1, SDN-2 and ODM approaches aims to modify genomic sequences in a way which can result in plants not containing any transgene, intragene or cisgene. Consequently, the GMO Panel concludes that those considerations which are specifically related to the presence of a transgene, intragene or cisgene included in section 4 and the conclusions of the Opinion on SDN-3 are not relevant to plants obtained via SDN-1, SDN-2 or ODM as defined in this Opinion. Overall, the GMO Panel did not identify new hazards specifically linked to the genomic modification produced via SDN-1, SDN-2 or ODM as compared with both SDN-3 and conventional breeding. Furthermore, the GMO Panel considers that the existing Guidance for risk assessment of food and feed from genetically modified plants and the Guidance on the environmental risk assessment of genetically modified plants are sufficient but are only partially applicable to plants generated via SDN-1, SDN-2 or ODM. Indeed, those guidance documents' requirements that are linked to the presence of exogenous DNA are not relevant for the risk assessment of plants developed via SDN-1, SDN-2 or ODM approaches if the genome of the final product does not contain exogenous DNA.

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“…Transfection of CRISPR/Cas components into protoplasts with subsequent regeneration of plants allowed to successfully introduce mutations with editing efficiencies ranging from 3% to 46%, resulting in either stable or transient expression in several plants including rice, soybean, A. thaliana , potato, grapevine, wheat, and lettuce [ 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 ]. This method allowed the creation of DNA-free edited plants by delivering preassembled Cas9-sgRNA ribonucleoproteins (RNPs) [ 79 , 80 , 82 ], which cannot be delivered by Agrobacterium [ 83 ]. The delivery of Cas9-sgRNA RNPs instead of plasmids that encode Cas9-sgRNA avoids that plasmids are degraded in cells by nucleases, resulting in small DNA fragments that may undesirably be inserted in the host genome [ 84 ].…”

Section: Delivery and Expression Of Crispr Systems In Plantsmentioning

confidence: 99%

Plant Viruses: From Targets to Tools for CRISPR

Varanda

Félix

Campos

et al. 2021

Viruses

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Plant viruses cause devastating diseases in many agriculture systems, being a serious threat for the provision of adequate nourishment to a continuous growing population. At the present, there are no chemical products that directly target the viruses, and their control rely mainly on preventive sanitary measures to reduce viral infections that, although important, have proved to be far from enough. The current most effective and sustainable solution is the use of virus-resistant varieties, but which require too much work and time to obtain. In the recent years, the versatile gene editing technology known as CRISPR/Cas has simplified the engineering of crops and has successfully been used for the development of viral resistant plants. CRISPR stands for ‘clustered regularly interspaced short palindromic repeats’ and CRISPR-associated (Cas) proteins, and is based on a natural adaptive immune system that most archaeal and some bacterial species present to defend themselves against invading bacteriophages. Plant viral resistance using CRISPR/Cas technology can been achieved either through manipulation of plant genome (plant-mediated resistance), by mutating host factors required for viral infection; or through manipulation of virus genome (virus-mediated resistance), for which CRISPR/Cas systems must specifically target and cleave viral DNA or RNA. Viruses present an efficient machinery and comprehensive genome structure and, in a different, beneficial perspective, they have been used as biotechnological tools in several areas such as medicine, materials industry, and agriculture with several purposes. Due to all this potential, it is not surprising that viruses have also been used as vectors for CRISPR technology; namely, to deliver CRISPR components into plants, a crucial step for the success of CRISPR technology. Here we discuss the basic principles of CRISPR/Cas technology, with a special focus on the advances of CRISPR/Cas to engineer plant resistance against DNA and RNA viruses. We also describe several strategies for the delivery of these systems into plant cells, focusing on the advantages and disadvantages of the use of plant viruses as vectors. We conclude by discussing some of the constrains faced by the application of CRISPR/Cas technology in agriculture and future prospects.

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The present and potential future methods for delivering CRISPR/Cas9 components in plants

Cited by 101 publications

References 94 publications

PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts

PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts

Applicability of the EFSA Opinion on site‐directed nucleases type 3 for the safety assessment of plants developed using site‐directed nucleases type 1 and 2 and oligonucleotide‐directed mutagenesis

Plant Viruses: From Targets to Tools for CRISPR

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