Transgenic Research

, Volume 28, Supplement 2, pp 61–64 | Cite as

DNA-free genome editing with preassembled CRISPR/Cas9 ribonucleoproteins in plants

  • Jongjin Park
  • Sunghwa ChoeEmail author
Proceedings Paper


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.


Functional genomics aims to discern the function of each gene in a genome of plants. To this end, plant scientists have depended on the mutants generated by random insertion of Agrobacterium T-DNA as a gene-tagging DNA in Arabidopsis and rice (Choe and Feldmann 1998). Inferring the function of the disrupted gene from the phenotypes of the T-DNA mutants facilitated plant functional genomics (Alonso et al. 2003). Despite substantial contribution of the random T-DNA mutants in understanding of the gene function, many of the genes are free of T-DNA insertions, and redundancy (Lloyd and Meinke 2012) of the plant genes in a genome hinders exposition of visible phenotype from a single mutant for functionally redundant genes, demanding novel methods of targeted disruption at multiplexed methods (Bak et al. 2011; Xu et al. 2009).

Insertion, deletion, and replacement of specific sequences in plant genome have been enabled by genome engineering tools including Zinc Finger Nuclease (ZFN) (Gaj et al. 2013), Transcription Activator-Like Effector Nuclease (TALEN) (Gaj et al. 2013), and Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 nuclease (CRISPR/Cas9) (Jinek et al. 2014) systems. ZFN, TALEN, and CRISPR/Cas9 each consist of two functional parts, one that directs the enzyme to a specific DNA sequence in the genome, and the other that functions as a DNA endonuclease.

Different from ZFN and TALEN, CRISPR–Cas9 RNA-guided endonucleases are directed to specific DNA sequences not by DNA-recognizing protein domains but by RNA complementary to a target sequences, specifically, a single-molecule guide RNA (gRNA) that is approximately 100 nucleotides long. Furthermore, the Cas9 protein harbors two endogenous nuclease subdomains, HNH and RuvC, thus abolishing the need for artificial linking to FoKI (Jinek et al. 2014) as is the case in ZFN and TALEN.

Because CRISPR–Cas9 is targeted to DNA in a mechanism that involves Watson Crick binding of sgRNA to protospacer DNA rather than protein, it is easier to design, synthesize, and incorporate the targeting molecule into the Cas9 nuclease apoprotein. The emergence of CRISPR–Cas9 has revolutionized the field of genome editing; indeed, a Google search using “CRISPR/Cas9” as a keyword resulted in 29.5 million hits as of August 2018, greatly increase relative to 48,000 hits as of April 2016. Given that the technology first appeared in 2012, it is fast becoming a routine technique. While CRISPR–Cas9 was first used to edit the genomes of viruses and prokaryotic cells, it is now being widely used in eukaryotic cells, including humans and plants.

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).
Fig. 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

Table 1

Comparison of different delivery methods


Way of CRISPR/Cas9 delivery







Mutation efficiency










Duration (weeks)





Foreign DNA integration





Antibiotic selection





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.


The possibility to edit plant genome in a DNA-free way raises the question of whether plants genetically edited with CRISPR–Cas9 fall within the scope of Genetically Modified Organisms (GMO) regulations. Many countries including the United States and Argentina designates CRISPR-edited plants as non-GMO (Waltz 2016) if foreign DNA encoding the Cas9 has been cured from the edited plants through segregation processes. Designation as non-GMO seeds could let seed innovators avoid the costs involved in obtaining ‘de-regulation’ status of their GMOs (Camacho et al. 2014). With the community acceptance of the CRISPR–Cas9 -edited plants as being equivalent to the seeds developed by conventional breeding programs greatly stimulate the seed developers especially small and medium sized companies to bring creative novel seeds relatively easy to market in shorter time. Novel seeds should include the plants with desirable traits, such as enhanced nutritional value, disease resistance, tolerance to abiotic stress, energy efficient architecture, and increased yield. This will eventually result in sustainable agriculture overall globally.



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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Naturegenic Inc.West LafayetteUSA
  2. 2.G+FLAS Life SciencesSeoulKorea
  3. 3.School of Biological SciencesCollege of Natural SciencesGwanak-gu, SeoulKorea

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