Genome Editing Technologies for Rice Improvement: Progress, Prospects, and Safety Concerns

Abstract

Rice (Oryza sativa) is an important staple food crop worldwide; to meet the growing nutritional requirements of the increasing population in the face of climate change, qualitative and quantitative traits of rice need to be improved. Stress-tolerant crop varieties must be developed with stable or higher yields under stress conditions. Genome editing and speed breeding have improved the accuracy and pace of rice breeding. New breeding technologies including genome editing have been established in rice, expanding the potential for crop improvement. Recently, other genome editing techniques such as CRISPR-directed evolution, CRISPR-Cas12a, and base editors have also been used for efficient genome editing in rice. Since rice is an excellent model system for functional studies due to its small genome and close syntenic relationships with other cereal crops, new genome-editing technologies continue to be developed for use in rice. In this review, we focus on genome-editing tools for rice improvement to address current challenges and provide examples of genome editing in rice. We also shed light on expanding the scope of genome editing and systems for delivering homology-directed repair templates. Finally, we discuss safety concerns and methods for obtaining transgene-free crops.

Keywords: CRISPR-Cas12a; CRISPR-Cas9; base editors; crop improvement; genome editing; rice; safety concerns; transgene-free.

Figure 1

Developing disease-resistant rice: Comparison of conventional breeding, genetic engineering and genome editing. (A) In conventional breeding there will be cross of a donor variety having disease resistance and commercial variety having high yield but susceptible to disease. The new variety developed in this way will be disease resistant with high yield. However, undesired genes from the donor variety will be incorporated along with the desired gene. (B) For developing a disease-resistant variety through genetic engineering, we need to isolate the desired gene from the donor variety and introduce into the commercial variety. This will result in a GMO crop. (C) In genome editing the susceptible gene will be targeted and disrupted. So, the new variety created in this way will be disease resistant with high yield.

Figure 2

Comparison of CRISPR/Cas9, CRISPR/Cas12a, and Base Editing. (A) In CRISPR/Cas9 system, Cas9 is a multicomponent protein and recognizes a canonical G-rich PAM at the 3' end of the target site. CRISPR RNA and trans-activating CRISPR RNAs are required to recruit Cas9. The Cas9 creates a DSB resulting in blunt ends. (B) In the CRISPR/Cas12a System, Cas12a is a single-component protein which recognizes T-rich PAM at the 5' end of the target sequence. No trans-activating CRISPR RNA is required. The DSB results in staggered cuts. (C) In base editing cytidine deaminase fused with dCas9 is used to target the desired location. There is no DSB, C is converted directly into U on the free strand, and during mismatch repair a C → T substitution can be created when the modified strand is used as template.

Figure 3

General strategy for improving rice through genome editing. The first step is gRNA design and construct development. After confirmation of the construct it is transferred either in Agrobacterium or coated on gold particles for biolistic bombardment. The construct is transformed to rice callus. The plants are regenerated from this callus. The regenerated plants are confirmed through PCR and restriction digestion, followed by sequencing. The plants are then screened for desired characteristics and moved to the greenhouse and then to field trials.

Figure 4

CRISPR-Cas mediated genome editing in rice for the improvement of different traits.

Figure 5

Development of transgene-free genetically edited rice plants for commercialization (A). The gRNA will be designed for targeted modification and during construct development appropriate transgene-free method will be chosen (e.g., transient expression of Cas9, RNP or suicide transgene method etc.) (B). For genome editing, the appropriate genome editing tool will be chosen depending upon the feasibility and type of modification that needs to be introduced. (C). The developed construct can be delivered to plant by Agrobacterium, biolistic or transfection. (D). Once the construct is delivered into the plant. It will perform its function by modifying the target site in the rice genome. After modification the transgene will be removed from the plants. (E). The regenerated plants will be screened for targeted modification without transgene. The plants developed in this way will be with desired traits without any transgene integration in the genome, so can be labeled as NON-GMO genetically edited plants by regulatory authorities.