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CURATOR

I am a PhD student working on tomato development at the John Innes Centre (Norwich, UK).

PINBOARD SUMMARY

Concerned about new CRISPR-edited crop varieties? Learn more about their safety and benefits!

New crop varieties generated by CRISPR/Cas9 genome editing (GE) have become a trend in modern agriculture, allowing the generation of a desired trait in a quick and targeted fashion. This technique is based on the use of the bacterial protein Cas9 to generate specific, targeted cuts in the plant genome that lead to mutations. However, some concerns have been raised about their impact in human health, as their development involves plant transformation techniques analogous to the ones used for development of GM crops.

So, are GE crops safe?

GE crops are GM-free. Although the Cas9 protein is required and must be introduced in the plant genome using GM techniques, offspring plants with the desired mutation can be selected without the Cas9 insertion, as these naturally segregate during plant reproduction. This means that all marketable GE crops are GM free.

Off-target effect. Another concern regarding GE crops is the lack of target-specificity during their development, which can produce other mutations different from the desired one. Off-target effects are routinely tested and are found in a very low rate, and, on top, more powerful computational and molecular tools are being developed to ensure that only well-characterised GE crops are released to the market.

CRISPR/Cas9 GE is equivalent to other accepted mutagenesis techniques. Researchers have used CRISPR to emulate known occurring mutations (for example, the rin tomato mutant). Equivalent phenotypes were observed, indicating that GE varieties are indistinguishable from traditionally generated mutants.

Then, what can CRISPR do for me?

GE varieties have been successfully generated for several vital crops, including tomato, rice or maize. The beneficial traits include resistant to disease pests, increase yield or resistant to abiotic stresses, among other desirable agronomical traits. Research is also aimed to increase benefits for the end-consumer (that’s you!), including health-promoting traits.

In addition, CRISPR use is spreading to other crops and its potential applications are increasing exponentially, with generation of new alleles by genetic shuffling being very promising.

Not only GE varieties are safe, but they can also be the solution of current problems in agriculture worldwide!

8 ITEMS PINNED

The CRISPR/Cas Genome-Editing Tool: Application in Improvement of Crops.

Abstract: The Clustered Regularly Interspaced Short Palindromic Repeats associated Cas9/sgRNA system is a novel targeted genome-editing technique derived from bacterial immune system. It is an inexpensive, easy, most user friendly and rapidly adopted genome editing tool transforming to revolutionary paradigm. This technique enables precise genomic modifications in many different organisms and tissues. Cas9 protein is an RNA guided endonuclease utilized for creating targeted double-stranded breaks with only a short RNA sequence to confer recognition of the target in animals and plants. Development of genetically edited (GE) crops similar to those developed by conventional or mutation breeding using this potential technique makes it a promising and extremely versatile tool for providing sustainable productive agriculture for better feeding of rapidly growing population in a changing climate. The emerging areas of research for the genome editing in plants include interrogating gene function, rewiring the regulatory signaling networks and sgRNA library for high-throughput loss-of-function screening. In this review, we have described the broad applicability of the Cas9 nuclease mediated targeted plant genome editing for development of designer crops. The regulatory uncertainty and social acceptance of plant breeding by Cas9 genome editing have also been described. With this powerful and innovative technique the designer GE non-GM plants could further advance climate resilient and sustainable agriculture in the future and maximizing yield by combating abiotic and biotic stresses.

Pub.: 06 May '16, Pinned: 23 Apr '17

Achieving Plant CRISPR Targeting that Limits Off-Target Effects

Abstract: The CRISPR-Cas9 system (clustered regularly interspaced short palindromic repeats with associated Cas9 protein) has been used to generate targeted changes for direct modification of endogenous genes in an increasing number of plant species; but development of plant genome editing has not yet fully considered potential off-target mismatches that may lead to unintended changes within the genome. Assessing the specificity of CRISPR-Cas9 for increasing editing efficiency as well as the potential for unanticipated downstream effects from off-target mutations is an important regulatory consideration for agricultural applications. Increasing genome-editing specificity entails developing improved design methods that better predict the prevalence of off-target mutations as a function of genome composition and design of the engineered ribonucleoprotein (RNP). Early results from CRISPR-Cas9 genome editing in plant systems indicate that the incidence of off-target mutation frequencies is quite low; however, by analyzing CRISPR-edited plant lines and improving both computational tools and reagent design, it may be possible to further decrease unanticipated effects at potential mismatch sites within the genome. This will provide assurance that CRISPR-Cas9 reagents can be designed and targeted with a high degree of specificity. Improved and experimentally validated design tools for discriminating target and potential off-target positions that incorporate consideration of the designed nuclease fidelity and selectivity will help to increase confidence for regulatory decision making for genome-edited plants.

Pub.: 01 Nov '16, Pinned: 23 Apr '17

ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions.

Abstract: Maize ARGOS8 is a negative regulator of ethylene responses. A previous study has shown that transgenic plants constitutively overexpressing ARGOS8 have reduced ethylene sensitivity and improved grain yield under drought stress conditions. To explore the targeted use of ARGOS8 native expression variation in drought tolerance breeding, a diverse set of over 400 maize inbreds was examined for ARGOS8 mRNA expression, but the expression levels in all lines were less than that created in the original ARGOS8 transgenic events. We then employed a CRISPR-Cas enabled advanced breeding technology to generate novel variants of ARGOS8. The native maize GOS2 promoter, which confers a moderate level of constitutive expression, was inserted into the 5'-untranslated region of the native ARGOS8 gene or was used to replace the native promoter of ARGOS8. Precise genomic DNA modification at the ARGOS8 locus was verified by PCR and sequencing. The ARGOS8 variants had elevated levels of ARGOS8 transcripts relative to the native allele and these transcripts were detectable in all the tissues tested, which was the expected results using the GOS2 promoter. A field study showed that compared to the WT, the ARGOS8 variants increased grain yield by 5 bushels per acre under flowering stress conditions and had no yield loss under well-watered conditions. These results demonstrate the utility of the CRISPR-Cas9 system in generating novel allelic variation for breeding drought tolerant crops. This article is protected by copyright. All rights reserved.

Pub.: 22 Jul '16, Pinned: 23 Apr '17

Efficient CRISPR/Cas9-based gene knockout in watermelon

Abstract: Abstract Key message CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Abstract Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon. Abstract Key message CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Key message CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Abstract Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon. AbstractGenome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.ClPDSClPDS

Pub.: 19 Dec '16, Pinned: 23 Apr '17