CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a family of DNA sequences in bacteria and archaea. The functions of CRISPR and CRISPR-associated (Cas) genes are essential in adaptive immunity in selected bacteria and archaea. It provides protection from viruses and other molecular parasites that can invade the bacterium and take over its genome.
In these systems, the immunity is mediated by a complex multi-protein molecular machinery that uses RNA molecules as molecular guides to recognize the invader and target it for destruction. Until now, it has been known that the complex itself has nuclease activity i.e., it can directly degrade the DNA and RNA of the invading viruses.
These repeats were initially discovered in the 1980s in E. coli, but their function wasn’t confirmed until 2007 by Barrangou and colleagues, who demonstrated that S. thermophilus can acquire resistance against a bacteriophage by integrating a genome fragment of an infectious virus into its CRISPR locus.
CRISPR-Cas9 is a tool in which, selected DNA from viruses or plasmids are designed and cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA – CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity.
Small RNAs (crRNA – CRISPR RNA) acts as a tag. And by editing this tag, scientists are able to target the enzyme to specific regions of DNA and make precise cuts, wherever they like. It has been used to ‘silence’ genes – effectively switching them off. When cellular machinery repairs the DNA break, it removes a small snip of DNA. In this way, researchers can precisely turn off specific genes in the genome.
This approach has been used previously to edit the HBB gene responsible for a condition called β-thalassaemia.
Recently, the researchers reported, in Frontiers in Plant Science (Front Plant Sci, 02 March 2018), the study results, which were thought to be the first to demonstrate the feasibility of using cutting-edge CRISPR technology to improve Theobroma cacao.
The cacao tree (Theobroma cacao), the source of cocoa, which grows in tropical regions, are the raw material of chocolate. Reliable productivity from cacao plants is essential to the multibillion-dollar chocolate industry, the economies of producing countries and the livelihoods of millions of smallholder cacao farmers. But it suffers significant losses to a variety of pathogens resulting in reduced incomes for millions of farmers in developing countries.
20-30 percent of cocoa pods destroyed preharvest, noted lead author Andrew Fister, postdoctoral scholar in plant science, College of Agricultural Sciences, Penn State.
Development of disease resistant cacao varieties is an essential strategy to combat this threat, but is limited by sources of genetic resistance and the slow generation time of this tropical tree crop.
Previous work in cacao identified a gene, known as TcNPR3 (Non-Expressor of Pathogenesis-Related 3 gene), which suppresses the plant’s disease response. The researchers hypothesized that using CRISPR-Cas9 to knock out this gene would result in enhanced disease resistance.
To test their hypothesis, they used Agrobacterium, a plant pathogen modified to remove its ability to cause disease and to introduce CRISPR-Cas9 components into detached cacao leaves and cotyledon cells. Subsequent analysis of treated tissue found deletions in 27 percent of TcNPR3 copies. When infected with Phytopthera tropicalis, a naturally occurring pathogen of cacao and other plants, the treated leaves showed greater resistance to the disease. The results suggested that the mutation of only a fraction of the copies of the targeted gene may be sufficient to trigger downstream processes, resulting in systemic disease resistance in the plant.
This study detected an increased resistance phenotype from the TcNPR3 mutation after mutagenizing on average 27% of TcNPR3 copies in the transiently transformed leaf tissue. The ability to detect CRISPR induced mutation phenotypes within a few days provided a powerful functional genomics tool and a way to assess the potential phenotype of any given genomic editing design prior recovery of mutagenized plants. The results indicate that in the case of major pathway regulators like NPR3, mutation of only a fraction of copies in a leaf may be sufficient to trigger downstream processes and result in a strong phenotypic change.
Researchers hypothesize that cells in which TcNPR3 is mutated by transient transformation signal to nearby cells, activates their defense responses. Induction of the defense pathway should lead to the downregulation of TcNPR3 throughout the leaf and induction of PR genes and other defense components, and the gene expression measurements were consistent with this prediction. The systemic acquired resistance pathway includes a complex network of signaling including NPR1 turnover, ROS burst, and epigenetic modification of defense genes.
In addition, the researchers also created CRISPR gene-edited cacao embryos, which they will grow into mature trees to test the effectiveness of this approach at a whole-plant level.
Future work will might investigate the molecular mechanisms connected to TcNPR3 knockout, and this work will be aided by further analysis of mature cacao trees harboring this TcNPR3 deletion.
Finally I would like to conclude that providing a new tool to accelerate breeding, CRISPR-Cas9 technology can help deliver insights into basic biology by offering a method to efficiently assess gene function and this study provides a ‘proof of concept’ that CRISPR-Cas9 technology can be a valuable tool in the effort to achieve these goals.
©BforBiotech by Bedadyuti Mohanty, Assistant Managing Editor by Profession and Bio-technologist by heart.