Can CRISPR improve rice, other crops?
With global climate change inching closer to crisis every year, researchers are working round the clock to leverage latest scientific strategies to improve stress tolerance in staple crops such as rice, to survive in harsh environments. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is one such research advance that scientists currently use to modify rice genomes to increase crop yield and survival. Rice is also the ideal model organism for studying editing strategies due to the small size of its genome and its shared genomic regions with other crops[i].
CRISPR-Cas9 genome editing
CRISPR-Cas9 (CRISPR associated protein) is an advanced genome editing strategy that can direct the insertion of a gene of interest into specific locations on the rice genome, called genomic safe harbors. These are regions on the host chromosome where new genes can be inserted without disrupting the function of other vital genes. In rice, the CRISPR element of the molecular toolkit guides Cas9 to such genomic safe harbors, where Cas9 cuts the DNA, making a double-stranded break (DSB).
When the cell’s DNA-repair machinery is triggered to mend the DSB, the gene of interest carried by the CRISPR-Cas9 is tailored into the host’s genome. Cells use two types of repair machinery for DSBs. Template-independent or non-homologous end-joining (NHEJ) repair ligates the ends of DNA together and does not need a donor template. However, NHEJ can cause random mutations or indels (insertions and deletions).
The repair machinery generally harnessed in the CRISPR-Cas9 approach is called the homology directed repair (HR), which is template-driven, precise, and mitigates the incidence of random indels of nucleotides at the insertion site. Cas9 requires a recognition motif on the host genome called protospacer adjacent motif (PAM) to make the DSB, but has been successfully employed by different research groups for genome editing in rice.
Figure 1: DNA repair machineries to repair double-stranded breaks. A: Components of the CRISPR-Cas9 module required for genome editing. B: The non-homologous end-joining repair mechanism introduces mutations in the host genome. C: The homology directed repair of DSBs that is precise and less error-prone than NHEJ.
CRISPR and genetically engineered rice
Conventional genome editing strategies use bacterium or particle guns to inject genes conferring desired traits into random regions on the host rice genome. Such random insertions may reduce overall yield. But with the help of CRISPR, scientists can precisely target a specific position on the rice genome and insert a gene that confers a desired trait.
Figure 2: Pipeline for production of gene-edited rice variants. A: Design of a guide RNA or CRISPR sequence and customized target gene for insertion. B: Choice of appropriate genome editing technique. C: Delivery of the CRISPR-Cas9 module into plant cells. D: Modification of the plant genome. E: Screening and selection of plant variants with desired traitsi.
For example, Golden Rice is a rice variant that owes its name to its bright yellow color, due to the presence of a genetically engineered insert for beta carotene. Beta carotene is the precursor for retinol (vitamin A), and fortifies nutritional intake of rice in areas of the world where there’s a dietary shortage of the vitamin. However, the full beta carotene gene cassette is a large piece of DNA fragment, averaging 5.2 kb. Usually, gene editing strategies limit insert sizes to less than 1.8 kb. They also require the presence of marker genes in the insert gene, to allow for the identification and selection of successfully modified variants. Such additional marker genes require extra regulatory approval for genetically modified crops (GMO), and may even cause concern in the general public regarding safe consumption.
With CRISPR, marker genes are not a necessity, thus facilitating the insertion of multiple genes with desired traits at a single locus on rice chromosomes.
Expanding genome editing strategies to improve crops
An improved CRISPR system in plants could help boost the function of up to seven genes at the same time. This kind of multiplexed activation can help scientists screen rice genomes to find genes that encode for beneficial traits, such as faster flowering or increased pathogen resistance. For such studies, the CRISPR-Cas9 module is inactivated so that it can only find and bind the targeted region on the genome. The DNA-bound module then recruits activator proteins to the target site, thus enhancing the expression of selective genes. This variant of the CRISPR-Cas9 shows promise for improved application and flexibility across many crops.
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 Mishra R, Joshi RK, Zhao K. Genome Editing in Rice: Recent Advances, Challenges, and Future Implications. Front Plant Sci. 2018;19(9):1361.
 Romero FM, Gatica-Arias A. CRISPR/Cas9: Development and application in rice breeding. Rice Sci 2019;26(5):265-281.
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