A Prospective Review on Selectable Marker-Free Genome Engineered Rice: Past, Present and Future Scientific Realm

Abstract

As a staple food crop, rice has gained mainstream attention in genome engineering for its genetic improvement. Genome engineering technologies such as transgenic and genome editing have enabled the significant improvement of target traits in relation to various biotic and abiotic aspects as well as nutrition, for which genetic diversity is lacking. In comparison to conventional breeding, genome engineering techniques are more precise and less time-consuming. However, one of the major issues with biotech rice commercialization is the utilization of selectable marker genes (SMGs) in the vector construct, which when incorporated into the genome are considered to pose risks to human health, the environment, and biodiversity, and thus become a matter of regulation. Various conventional strategies (co-transformation, transposon, recombinase systems, and MAT-vector) have been used in rice to avoid or remove the SMG from the developed events. However, the major limitations of these methods are; time-consuming, leftover cryptic sequences in the genome, and there is variable frequency. In contrast to these methods, CRISPR/Cas9-based marker excision, marker-free targeted gene insertion, programmed self-elimination, and RNP-based delivery enable us to generate marker-free engineered rice plants precisely and in less time. Although the CRISPR/Cas9-based SMG-free approaches are in their early stages, further research and their utilization in rice could help to break the regulatory barrier in its commercialization. In the current review, we have discussed the limitations of traditional methods followed by advanced techniques. We have also proposed a hypothesis, "DNA-free marker-less transformation" to overcome the regulatory barriers posed by SMGs.

Keywords: clustered regularly interspaced short palindromic repeats/crispr associated Cas9 (Crispr/Cas9); genetic engineering; genetically modified (GM) -regulation; rice; selectable marker genes (SMGs).

FIGURE 1

Status of selectable markers used for the generation of SMG-free transgenic rice. Representation of various selectable markers contribution (A), Timeline representation of SMG-free techniques used in rice (B), Proportion of different molecular approaches in developing SMG-free engineered rice (C).

FIGURE 2

A schematic model of the CRISPR-based RNP method. The model summarizes the use of various explants (protoplast, embryo, zygote, and callus) and the protocol used for genome editing to produce SMG-free transgenic rice. RNP, (Ribonucleoprotein) complex; PEG, (Polyethylene glycol); RED, (Restriction enzyme digestion).

FIGURE 3

Editing of OsBADH2 gene for generation of aromatic rice using RNP approach. Acclimatized T₀ edited plants for OsBADH2 gene grown under glasshouse conditions (A), Detection of RNP-based editing in the T₀ generation through mutation site based specific primers technique (MSBSP). Encircled lane depicts the mutation (B), A electropherogram showing the result of Sanger sequencing (C), Multiple sequence alignment of putative T₀ plants showing the addition of a nucleotide “A” 4-bp upstream of the PAM site (D), The ORF of OsBADH2 exon seven in PR114 (E), The ORF of Osbadh2 exon seven in the edited plant, 11-4 showing change in the last four amino acid sequences indicating the disruption of protein chain (F).

FIGURE 4

A hypothetical model for the development of DNA and marker-free genome-edited plants.