Aug. 7, 2019
Processes of traditional trait development in plants depend on genetic variations derived from spontaneous mutation or artificial random mutagenesis. Limited availability of desired traits in crossable relatives or failure to generate the wanted phenotypes by random mutagenesis led to develop innovative breeding methods that are truly cross-species and precise. To this end, we devised novel methods of precise genome engineering that are characterized to use pre-assembled CRISPR/Cas9 ribonucleoprotein (RNP) complex instead of using nucleic ands or Agrobacterium. We found that our methods successfully engineered plant genomes without leaving any foreign DNA footprint in the genomes. To facilitate introduction of RNP into plant nucleus, we first obtained protoplasts after removing the transfection barrier, cell wall. Whole plants were regenerated from the single cell of protoplasts that has been engineered with the RNP. Pending the improved way of protoplast regeneration technology especially in crop plants, our methods should help develop novel traits in crop plants in relatively short time with safe and precise way.
Different methods of CRISPR–Cas9 delivery in plants
Genes encoding CRISPR–Cas9 components have successfully been expressed both stably and transiently in plants. Multiple targets can simultaneously be edited when several sgRNAs are expressed in one cell. Considering that redundant genes are common in the plant genome, CRISPR–Cas9-mediated multiplexed genome editing could yield higher-order mutants with relative ease compared to conventional crossing methods.
Instead of directly introducing the DNA plasmids encoding CRISPR–Cas9 effector protein and sgRNA, each of the CRISPR–Cas9 components can be separately prepared and assembled in vitro and subsequently transfected into lettuce protoplasts for genome editing (Woo et al. 2015). The regenerated plants originating from a single engineered protoplast were shown to inherit the mutation in a Mendelian fashion. In contrast to the plasmid-based system, off-target effects were negligible, possibly due to the short life-time of the introduced CRISPR–Cas9 complex. However, when Cas9 is administered as DNA, the functional enzymes are made continuously and they could increase the possibility of off-target effects. The Fig. 1 illustrates different ways of CRISPR–Cas9 genome editing in plants (Table 1).
Different methods of CRISPR–Cas9 delivery into plant cells. Transgenic method includes Agrobacterium-mediated transfer of T-DNA encoding Cas9 protein and sgRNA into plant cell. Non-transgenic ways include transfection of plasmid, mRNA-sgRNA, and ribonucleoprotein (RNP) into callus or protoplasts. Subsequent regeneration of whole plants from transfected cells results in genome-engineered plants
Transgenic and transient methods are compared in terms of different categories. Of these, RNP stands out in efficiency, specificity, and the nature of non-transgenesis
Although transfection of CRISPR–Cas9 sgRNA-protein complex, ribonucleoprotein (RNP), into callus via biolistic bombardment and subsequent regeneration of plants out of the transformed calli is considered an alternative to protoplast-based genome editing, callus-based methods harbor multiple problems like chimeric tissues consisting of genome-edited and non-edited cells. Subsequent genetic fixation into a monogenic line should follow for stable inheritance of the edited traits. Currently, many of plants are available for regeneration of whole plants from protoplasts. These include Chlamydomonas (Baek et al. 2016), petunia (Subburaj et al. 2016), wheat (Liang et al. 2017), maize (Svitashev et al. 2016), apple (Malnoy et al. 2016), and soybean (Kim et al. 2017), and the list seems to expand because of the merits of the RNP technology.
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