Protecting plants from viral pathogens is a major challenge faced by the agricultural industry. Pathogenic infections can potentially threaten global food security by severely affecting crop yields. In order to combat prevalent plant diseases, versatile genome editing technologies such as CRISPRCas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated protein 9), have been employed to drastically accelerate resistant plant species breeding.
Based on a dynamic immune system from Streptococcus pyogenes, the CRISPRCas9 system is a complex of two components: a Cas9 endonuclease and single-guide RNA (sgRNA). Cas9 performs double-stranded breaks on target DNA, and the single-guide RNA (sgRNA) directs Cas to target sequences. Induced double-stranded breaks by Cas9 are mainly repaired by non-homologous end-joining, which often results in deleterious sequence insertions or deletions. Mutagenesis may alter open reading frames (ORF's) or may cause protein truncation with the introduction of a premature stop codon. Loss of protein function by CRISPRCas9 makes it an appropriate tool for both viral genome and plant genome modification.
CRISPRCas9 machinery can be used to modify the genetic material in viruses. For example, integrated endogenous Banana streak viruses (eBSV) pose a great challenge for banana plant breeding programmes. In a study, targeted mutagenesis by CRISPR knocked out three eBSV protein open reading frames (ORF's) in Gonja Manjaya plants, preventing the formation of functional BSV viral proteins and pathogenic phenotypes e.g., yellowed, and blackened leaves. sgRNA's were generated for each of the ORF sequences and integrated into a Cas9 expression vector containing a kanamycin resistance selective gene. Agrobacterium-mediated, electroporation of the plasmid transformed the banana plant embryogenic cell suspensions, and both wild-type controls and selected mutants were planted. Both groups were subjected to water stress and observed daily for the development of BSV symptoms. eBSV was activated and disease phenotypes appeared in all the control plants, whereas the modified plants remained asymptomatic under the same conditions. PCR showed that asymptomatic plants contained mutations in all the targeted ORF sites, where CRISPR was able to permanently silent eBSV. Due to its high efficacy, CRISPR could be used to inactivate other endogenous viral genomes for effective disease prevention in the future.
Viral proliferation can also be impeded through targeted mutagenesis of sequences in host genomes encoding vital replication machinery. In cucumber plants, CRISPRCas9 has been utilised to silence host genes needed for infection by Cucumber vein yellowing virus (CVYV), Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot mosaic virus-W (PRSV-W).
Viruses require an eIF4E translation initiation factor complex for protein translation. Protein factors in the complex bind to the 5' m7G cap and 3' poly a tail of viral mRNA, through a 5' viral-encoded protein (VPg). In this study, disruption of the covalent interaction between the VPg protein and the host factor complexes was achieved through CRISPRCas9 targeted mutagenesis of the recessive eIF4E gene. Two sgRNAs were constructed to target two exons in the eIF4E gene coding region. Transgenic lines were synthesised by Agrobacterium-mediated transformation, and the plants were selected for by PCR and kanamycin exposure. Sequencing showed that there were a variety of deletion and insertion indels at the two targeted sites of the transformed plants.
The study went on to cross-pollinate these transgenic mutant seeds with wild-type 'Bet Alfa' seeds, which produced progeny with Mendelian genotypes: 1:2:1 (homozygous: heterozygous: wild-type). Sequencing demonstrated that there was independent segregation of the transgene Cas9sgRNA and the mutant eiF4E gene. Since this meant they were on different chromosomes, it was possible to select non-transgenic plants and use them in generational crosses to synthesise non-transgenic, mutant, homozygous progeny. The viral susceptibilities of these plants were then compared with heterozygous, and wild-type plants under the exposure of viral pathogens. The homozygous genotype conferred immunity to CVYV and resistance to ZYMV and PRS, however heterozygous or wild-type plants, were highly vulnerable to all three diseases. Since the resistant plants were non-transgenic, they had no impact on plant development, were safe for human consumption and release into the environment. This experiment showed that it was possible to produce non-transgenic, virus-resistant plants using CRISPRCas9. Future work could involve the utilisation of this gene-editing technology to make modifications to a wide variety of plant genomes, for protection against other viral pathogens.
CRISPRCas9 technology is an example of a highly effective genome-editing technology, however, there are major issues with off-target mutagenesis, caused by a high tolerance for sgRNA sequence mismatches, and protracted expression of Cas9 nuclease. To prevent this, utilisation of virus-induced promotors, instead of constitutive promoters for the expression of Cas9, would be effective for the regulated and induced expression of the CRISPRCas9 system. Moreover, the technology has shown poor plant cell delivery efficacy, and it is essential for future work to consider how optimal CRISPRCas9 delivery can be achieved.
Moreover, CRISPR-resistant viruses may arise through the direct targeting of viral genomes, Cas9 cleavage, and subsequent strand reformation. Variant formation may lead to rapid viral evolution and should be avoided through simultaneous targeting of many viral genome sequences. Furthermore, CRISPR-based nickase enzymes could be utilised to create larger deletions in the viral genome, thereby decreasing strand recombination frequency.
To conclude, plant viruses pose serious problems for the agricultural industry, and CRISPRCas9 offers a novel approach for the development of resistant plant species. Genome modifications made to both plant and viral genomes have been shown to drastically reduce viral infection susceptibility. There are, however, issues with off-target mutagenesis, formation of CRISPR-resistant virus species and effective plant cell delivery. Further work must be done to enhance this gene-editing technology, for optimal plant protection and agricultural progression.
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