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Figure 1. 

Schematic diagram for the wet-lab workflow of CRISPR/Cas-based genome editing, showing the limitations of current transformation and regeneration protocols. After CRISPR/Cas reagents, such as a plasmid DNA vector or RNP for the genes of interest, are transformed into explant cells, these cells must then be regenerated into mutant transgenic plants. Each step of the tissue culture process reduces its efficiency owing to the regeneration of non-transformed plants and the regeneration of transformed plants that lack the desired edit. Antibiotics are typically added to the culture medium to increase the proportion of transformed cells by inhibiting the growth of non-transformed cells (only in the Agrobacterium T-DNA transfer method). The transformation efficiency, regeneration rate, and in vivo activity of CRISPR/Cas reagents all impact the total genome editing efficiency during this process. However, genome editing efficiency in most tree genome editing practices (Table 1) has not been accurately measured. Efficiency is typically calculated as A/(A+B+C), where A indicates the number of mutant transgenic plants, and B and C indicate the numbers of non-mutant transgenic plants obtained from transformed and non-transformed cells. This does not account for the number of explant cells that were transformed but not regenerated. Most tree genome editing studies have focused more on whether the CRISPR/Cas reagents function than on their efficiency. Conventional protocols for transformation and regeneration are laborious and time-consuming, and their low efficiencies are major obstacles to tree genome editing using the CRISPR/Cas system. Possible solutions to these problems are discussed in Section 4.

Figure 2. 

Direct delivery of CRISPR/Cas reagents (plasmid or RNP) and potential barriers affecting their delivery efficiency and intranuclear genome editing activities. The active form of CRISPR/Cas reagents is the RNP, which is generated from transcription and translation of the CRISPR/Cas and sgRNA sequences. Because transcription only takes place in the nucleus, these plasmids must therefore gain entry to this cellular compartment. In the nucleus, Cas+NLS and sgRNAs are transcribed into RNAs, and the Cas + NLS mRNA must be exported into the cytoplasm to be translated into the Cas + NLS protein, which then re-enters the nucleus to form the RNP complex with sgRNAs. Therefore, the plasmid delivery process involves a total of three passes through the nuclear envelope. Although this process has been studied extensively, it still remains unclear how the nuclear envelope regulates the import of plasmid DNA, RNA, or RNP complexes into the nucleus, and the low efficiency of direct delivery systems may be due to the negative regulatory role of the nuclear envelope during the nucleocytoplasmic transfer of CRISPR/Cas reagents into the nucleus. Furthermore, intracellular protein and RNA degradation systems, such as the Ubiquitin-Proteosome and RNA exosome, may be potential obstacles for the RNP complex. These “degradosomes” may render the activity of RNPs more transient, resulting in a much lower editing efficiency.

Figure 3. 

Faster and easier regeneration of genome-edited plants by tissue culture–independent protocols. (a) Conventional tissue culture is both tedious and laborious. This process normally takes anywhere from six to eighteen months and requires a sterile environment and a large amount of tissue culture medium, dishes, bottles, and chemical reagents. Its regeneration efficiency is relatively low, and recalcitrancy limits its utility. (b) Recently, novel technologies, such as mobilization of sgRNAs by FT mRNA fusion and de novo meristem induction, have been developed, enabling researchers to overcome some of the problems of conventional tissue culture. In the FT mRNA/sgRNAs protocol, FT mRNA encodes the mobile florigen essential for induction of flowering, which is fused to sgRNAs to facilitate their movement from the leaf to the shoot apical meristem. This causes genome editing of the floral meristem, which results in genome-edited seed production. In the de novo meristem induction protocol, genome editing and meristem induction are performed simultaneously to generate genome-edited seeds. These in planta transformation protocols require only one or two months to generate genome-edited plants. In addition, these protocols do not require laborious processes of sterilization and sterile tissue culture.