The CRISPR revolution in plant disease management
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Ngangbam Punita and Snata Kaushik
Plant disease has always been a nuisance in food production. Since then, it has always been a challenge for plant pathologists to come up with effective and sustainable management strate- gies. Theoretically, the use of resistant cultivars is one of the most effective ways to reduce the negative impacts by plant pathogens. However, in nature it is not assured that we will obtain a resistant trait to a particular disease. Even if the gene conferring the resistance is found in nature, it takes years long process to incorporate it in cultivated varieties. For all, this hitch came to an end with the discovery of genome editing through CRISPR in 2012. There are no biologists who have not heard about CRISPR. Just 4 years ago, the Nobel Prize in Chemistry was awarded for the discovery of CRISPR/Cas9 genome editing technology by Jennifer Doudna and Emmanuelle Charpentier. Yet, very few of the general public are aware of this ground-breaking technology. So, what exactly is CRISPR technology and how is it applicable in plant disease management?
Clustered Regularly Interspaced Palindromic Repeats (CRISPR) is an adaptive immunity found in bacteria and archaea to defend themselves against invading viruses through targeted cleavage of foreign nucleic acid. Basically, this defense mechanism comprises of DNA sequences of different invading viruses that has been incorporated into the bacterial genome separated by interspaced repeats and a nuclease protein called Cas protein that cleaves the target DNA. The mechanism behind CRISPR involves cleavage of the invading viral DNA and incorporating this DNA sequence into the bacterial genome which in turn serves as a memory to the bacteria and in subsequent infection by the same virus, the CRISPR repeats transcribed into a guide RNA (gRNA) guides the Cas9 to the target site to cleave the viral DNA making itself immune. The discovery of this technology has revolutionized the field of biotechnology, biomedicine and agriculture.
CRISPR/Cas technology can be employed when we can identify which genes to alter and what alteration is to be made to the particular gene to make the plant resistant. Disease resistance can be imparted by manipulation of the susceptible gene (S gene) or negative regulators of plant immunity. Scientist artificially constructs a DNA sequence that encode for a gRNA specific for a particular DNA sequence and a complimentary Cas protein. The construct is then incorporated into the plant where it gets transcribed and translated into the gRNA and Cas protein. The gRNA goes and binds to the target site and the Cas protein knocks out the target by causing a double strand break. Once the susceptibility gene in the plant is knocked out, the pathogen fails to infect or proliferate on the host thus making the host plant resistant. However, one should keep in mind that the target gene should not be linked to other vital function for normal functioning of the plant. With recent advancements, three to four gRNAs can be cloned for multiple genetic manipulation. The resounding achievement of CRISPR/Cas9 in plant genetics has influenced the utilization of this technology to develop durable broad spectrum resistance to plant pathogens.
There are several examples for successfully conferring disease resistance to fungal, bacterial and viral plant pathogens through CRISPR technology. The targeted mutation of the mildew resistance locus O (MLO), a conserved S gene in wheat and tomato leads to powdery mildew fungal pathogen resistance. In rice, successful editing of the OsSWEET14, OsSWEET13 and OsSWEET11 susceptible genes by CRISPR/Cas9 led to the resistance against multiple races of bacterial blight pathogen, Xantho-monasoryzae. CRISPR/Cas9 machinery has also been successfully utilized to deactivate endogenous Banana Streak Virus, a double stranded DNA virus when stably integrated into the B genome of plantain (AAB), reducing a big challenge in breeding and dissemination of resistant hybrids. Other than the gene knockdown function, scientist also use CRISPR/Cas system to incorporate resistant gene (R gene) available in wild types into cultivated varieties.
Alternatively, multiplex genome editing may be performed on elite commercial cultivars to engineer and stack different R alleles and defense-related genes for broad-spectrum and durable disease resistance.
Some limitations with this technology include the classification of the plants as transgenic, potential non-specific cleavage by the Cas nuclease, and the evolution of viral resistance to the targeting gRNA. Government regulations and public concerns with genetically modified organisms (GMOs) are important issues associated with prac- tical application and commercialization of CRISPR/Cas-edited crops across the globe.
The US Department of Agriculture has determined that certain genome- edited crops will not be regulated as GMOs, if the genetic modification involves deletion of DNA fragment, substitution of single nucleotide that could otherwise result from chemical or radiation mutagenesis, or insertion of DNA segment that could be achieved via traditional breeding with a sexually compatible species.
(To be contd)
Plant disease has always been a nuisance in food production. Since then, it has always been a challenge for plant pathologists to come up with effective and sustainable management strate- gies. Theoretically, the use of resistant cultivars is one of the most effective ways to reduce the negative impacts by plant pathogens. However, in nature it is not assured that we will obtain a resistant trait to a particular disease. Even if the gene conferring the resistance is found in nature, it takes years long process to incorporate it in cultivated varieties. For all, this hitch came to an end with the discovery of genome editing through CRISPR in 2012. There are no biologists who have not heard about CRISPR. Just 4 years ago, the Nobel Prize in Chemistry was awarded for the discovery of CRISPR/Cas9 genome editing technology by Jennifer Doudna and Emmanuelle Charpentier. Yet, very few of the general public are aware of this ground-breaking technology. So, what exactly is CRISPR technology and how is it applicable in plant disease management?
Clustered Regularly Interspaced Palindromic Repeats (CRISPR) is an adaptive immunity found in bacteria and archaea to defend themselves against invading viruses through targeted cleavage of foreign nucleic acid. Basically, this defense mechanism comprises of DNA sequences of different invading viruses that has been incorporated into the bacterial genome separated by interspaced repeats and a nuclease protein called Cas protein that cleaves the target DNA. The mechanism behind CRISPR involves cleavage of the invading viral DNA and incorporating this DNA sequence into the bacterial genome which in turn serves as a memory to the bacteria and in subsequent infection by the same virus, the CRISPR repeats transcribed into a guide RNA (gRNA) guides the Cas9 to the target site to cleave the viral DNA making itself immune. The discovery of this technology has revolutionized the field of biotechnology, biomedicine and agriculture.
CRISPR/Cas technology can be employed when we can identify which genes to alter and what alteration is to be made to the particular gene to make the plant resistant. Disease resistance can be imparted by manipulation of the susceptible gene (S gene) or negative regulators of plant immunity. Scientist artificially constructs a DNA sequence that encode for a gRNA specific for a particular DNA sequence and a complimentary Cas protein. The construct is then incorporated into the plant where it gets transcribed and translated into the gRNA and Cas protein. The gRNA goes and binds to the target site and the Cas protein knocks out the target by causing a double strand break. Once the susceptibility gene in the plant is knocked out, the pathogen fails to infect or proliferate on the host thus making the host plant resistant. However, one should keep in mind that the target gene should not be linked to other vital function for normal functioning of the plant. With recent advancements, three to four gRNAs can be cloned for multiple genetic manipulation. The resounding achievement of CRISPR/Cas9 in plant genetics has influenced the utilization of this technology to develop durable broad spectrum resistance to plant pathogens.
There are several examples for successfully conferring disease resistance to fungal, bacterial and viral plant pathogens through CRISPR technology. The targeted mutation of the mildew resistance locus O (MLO), a conserved S gene in wheat and tomato leads to powdery mildew fungal pathogen resistance. In rice, successful editing of the OsSWEET14, OsSWEET13 and OsSWEET11 susceptible genes by CRISPR/Cas9 led to the resistance against multiple races of bacterial blight pathogen, Xantho-monasoryzae. CRISPR/Cas9 machinery has also been successfully utilized to deactivate endogenous Banana Streak Virus, a double stranded DNA virus when stably integrated into the B genome of plantain (AAB), reducing a big challenge in breeding and dissemination of resistant hybrids. Other than the gene knockdown function, scientist also use CRISPR/Cas system to incorporate resistant gene (R gene) available in wild types into cultivated varieties.
Alternatively, multiplex genome editing may be performed on elite commercial cultivars to engineer and stack different R alleles and defense-related genes for broad-spectrum and durable disease resistance.
Some limitations with this technology include the classification of the plants as transgenic, potential non-specific cleavage by the Cas nuclease, and the evolution of viral resistance to the targeting gRNA. Government regulations and public concerns with genetically modified organisms (GMOs) are important issues associated with prac- tical application and commercialization of CRISPR/Cas-edited crops across the globe.
The US Department of Agriculture has determined that certain genome- edited crops will not be regulated as GMOs, if the genetic modification involves deletion of DNA fragment, substitution of single nucleotide that could otherwise result from chemical or radiation mutagenesis, or insertion of DNA segment that could be achieved via traditional breeding with a sexually compatible species.
(To be contd)