Next Article in Journal
Identification of Pathways Associated with Placental Adaptation to Maternal Nutrient Restriction in Sheep
Next Article in Special Issue
Genomic Analysis of Selected Maize Landraces from Sahel and Coastal West Africa Reveals Their Variability and Potential for Genetic Enhancement
Previous Article in Journal
Biomarkers, Master Regulators and Genomic Fabric Remodeling in a Case of Papillary Thyroid Carcinoma
Previous Article in Special Issue
Development of Chromosome Segment Substitution Lines (CSSLs) Derived from Guangxi Wild Rice (Oryza rufipogon Griff.) under Rice (Oryza sativa L.) Background and the Identification of QTLs for Plant Architecture, Agronomic Traits and Cold Tolerance
Article

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

1
CropScience Department, Federal University of Santa Catarina, Florianópolis 88034000, Brazil
2
GenØk—Centre for Biosafety, Siva innovasjonssenter Postboks 6418, 9294 Tromsø, Norway
*
Author to whom correspondence should be addressed.
Received: 18 August 2020 / Revised: 28 August 2020 / Accepted: 30 August 2020 / Published: 2 September 2020
(This article belongs to the Special Issue Recent Advances in Genetics and Breeding of Major Staple Food Crops)
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. View Full-Text
Keywords: gene editing; mutagenesis; genetically modified; genetically modified organism (GMO); crop breeding; ribonucleoprotein complex (RNP); genetic screening gene editing; mutagenesis; genetically modified; genetically modified organism (GMO); crop breeding; ribonucleoprotein complex (RNP); genetic screening
Show Figures

Figure 1

MDPI and ACS Style

Sant’Ana, R.R.A.; Caprestano, C.A.; Nodari, R.O.; Agapito-Tenfen, S.Z. PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts. Genes 2020, 11, 1029. https://0-doi-org.brum.beds.ac.uk/10.3390/genes11091029

AMA Style

Sant’Ana RRA, Caprestano CA, Nodari RO, Agapito-Tenfen SZ. PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts. Genes. 2020; 11(9):1029. https://0-doi-org.brum.beds.ac.uk/10.3390/genes11091029

Chicago/Turabian Style

Sant’Ana, Rodrigo R.A., Clarissa A. Caprestano, Rubens O. Nodari, and Sarah Z. Agapito-Tenfen 2020. "PEG-Delivered CRISPR-Cas9 Ribonucleoproteins System for Gene-Editing Screening of Maize Protoplasts" Genes 11, no. 9: 1029. https://0-doi-org.brum.beds.ac.uk/10.3390/genes11091029

Find Other Styles
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop