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

Abstract

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.

Efficient Targeted Mutagenesis Mediated by CRISPR-Cas12a Ribonucleoprotein Complexes in Maize

“…One possible explanation is that the presence of a more open chromatin structure for the MIR604TS2 target locus in leaf vs. immature embryos. It should be noted that protoplast transfection has been widely applied to assess Cas9 RNP activity against different targets in various plant species including maize (Woo et al, 2015;Malnoy et al, 2016;Svitashev et al, 2016;Andersson et al, 2018;Liang et al, 2018;Lin et al, 2018;Murovec et al, 2018;Sant'Ana et al, 2020). Since a much higher percentage of cells can receive RNP in comparison with direct embryo bombardment, protoplast transfection is expected to result (on average) in much higher editing efficiencies.…”

CRISPR ribonucleoprotein-mediated genetic engineering in plants

“…Efficiencies of different Cas systems and gRNAs can be quickly evaluated using this method. PEG-mediated protoplast assays have been developed for many plant species, including Arabidopsis thaliana , rice ( Oryza sativa ), lettuce ( Lactuca sativa ) ( Woo et al., 2015 ), tobacco ( Nicotiana tabacum and N. attenuata ) ( Woo et al., 2015 ; Kim et al., 2017 ), petunia ( Petunia × hybrida ) ( Subburaj et al., 2016 ; Yu et al., 2020 ), maize ( Zea mays ) ( Sant’Ana et al., 2020 ), grapevine ( Vitis vinifera ), apple ( Malus × domestica ) ( Malnoy et al., 2016 ), wheat ( Triticum aestivum ) ( Liang et al., 2017 ), soybean ( Glycine max ) ( Kim et al., 2017 ; Kim and Choi, 2020 ), potato ( Solanum tuberosum ) ( Andersson et al., 2018 ; González et al., 2020 ), cabbage ( Brassica oleracea ), Chinese cabbage ( Brassica rapa ) ( Murovec et al., 2018 ), and banana ( Musa spp.) ( Wu et al., 2020 ).…”

“…Chardonnay) Mildew locus O 7 (MLO-7) Cas9 protoplast PEG targeted deep sequencing 0.1% Malnoy et al. (2016) Maize ( Zea mays ) Inositol phosphate kinase (IPK) Cas9 protoplast PEG Sanger sequencing 0.85%–5.85% Sant’Ana et al. (2020) Hot pepper ( Capsicum annuum cv.…”

DNA-free gene editing in plants: a brief overview

“…Soon after the first successful reports [9], the DNA-free editing approach was applied on a number of species. Since the review of Metje-Sprink et al [8], researchers have achieved DNA-free editing in Nicotiana benthaminiana [10], potato [11], wheat and maize [12,13], Brassicaceae [14], rice [15], banana [16], lettuce [17], pepper [18]. While two years is not a significantly long period, it is clear that commercially important species have become the main target for DNA-free genomic manipulations (i.e.…”

“…Ribonucleoprotein complexes and nanoparticles are delivered in plant cells mostly by particle bombardment [23] or protoplast transformation. In the latter case lipofection [14,24] or PEG-Ca 2þ transfection [13,15,16] is used. Electroporation is another way to deliver macromolecules across protoplast membrane and well-designed protocols are available [7], but there have been no reports (to our knowledge) during the last two years.…”