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

Khatodia S, Bhatotia K, Passricha N, Khurana SM, Tuteja N - Front Plant Sci (2016)

Bottom Line: The Clustered Regularly Interspaced Short Palindromic Repeats associated Cas9/sgRNA system is a novel targeted genome-editing technique derived from bacterial immune system.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.

View Article: PubMed Central - PubMed

Affiliation: Amity Institute of Biotechnology, Amity University Haryana Gurgaon, India.

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.

No MeSH data available.


Related in: MedlinePlus

The generation of transgene free genetically edited (GE) crops. The transgene free homozygous mutants with desired genetic modifications at the targeted loci and without RGEN transgene construct could be selected by selfing of GE0 generation plants and after segregation of the transgene in the next GE1 generation. The GE plants could be selected by PCR/RE genotyping and DNA sequencing of clones and negatively selecting for the transgene free plants with desired modification in the first generation only.
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Figure 5: The generation of transgene free genetically edited (GE) crops. The transgene free homozygous mutants with desired genetic modifications at the targeted loci and without RGEN transgene construct could be selected by selfing of GE0 generation plants and after segregation of the transgene in the next GE1 generation. The GE plants could be selected by PCR/RE genotyping and DNA sequencing of clones and negatively selecting for the transgene free plants with desired modification in the first generation only.

Mentions: Routine targeted mutagenesis in plants by the CRISPR/Cas9 system will definitely help open up new dimensions in plant biology research. The conventional random mutagenesis approaches were not able to access every gene for inactivation study because of the random nature of the gene integration. CRISPR-Cas9 technology would be of great help in generating mutants for inaccessible genes, mutate multiple loci and generate large deletions, which can, therefore, accelerate plant breeding without actually introducing a transgene (Figure 4). Although, genetically modified crops could have been a solution for crop improvement, if the controversies concerning the probable environmental and health implications of GM crops could be avoided (Hilbeck et al., 2011). Using the technique of genetic engineering a DNA construct can be directly inserted into one or more chromosomes in a random manner for manipulation of the genome. However, the random gene insertions can have undesirable effects and are not favorable for making large intensive changes, such as in case of adding an entire metabolic pathway into a plant (Lau et al., 2014). The plant breeding techniques have been used regularly to introduce new traits into cultivated varieties using existing natural genetic variation and random mutagenesis. The CRISPR/Cas system introduces precise modifications into a plant genome, which inherits stably and transgene region could be removed simply after the target gene editing to make transgene free plants during crop variety improvement (Mahfouz et al., 2014; Gao and Zhao, 2014b; Kanchiswamy et al., 2015; Xu et al., 2015; Figure 5). The Cas9/sgRNA system, is now one of the new plant breeding techniques (NPBT) along with the ZFNs (Zinc Finger Nucleases) and TALENs (Transcription activator like effector nucleases) like genome editing technologies. The NPBTs are faster than traditional breeding methods and can produce a segregant line that notably lacks the transgenic insert (Podevin et al., 2012; Araki and Ishii, 2015; Schaart et al., 2015; Woo et al., 2015). The plants developed by NPBTs are identical to the classically bred plants and these should be evaluated according to the resulting end product rather than the process of creation (Giddings et al., 2012; Hartung and Schiemann, 2014; Woo et al., 2015). Thus, in such a case CRISPR edited plants might be out of the current GMO regulations, but still this pose new challenges for the regulation and social acceptance of genetically edited (GE) crops (Voytas and Gao, 2014; Araki and Ishii, 2015; Jones, 2015; Kanchiswamy et al., 2015). NPBTs are currently in debate by advisory and regulatory authorities in relation to the GMO legislation to classify GE crop varieties produced using genome editing as non-GM for the social acceptance (Lusser et al., 2012; Voytas and Gao, 2014; Araki and Ishii, 2015).


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

Khatodia S, Bhatotia K, Passricha N, Khurana SM, Tuteja N - Front Plant Sci (2016)

The generation of transgene free genetically edited (GE) crops. The transgene free homozygous mutants with desired genetic modifications at the targeted loci and without RGEN transgene construct could be selected by selfing of GE0 generation plants and after segregation of the transgene in the next GE1 generation. The GE plants could be selected by PCR/RE genotyping and DNA sequencing of clones and negatively selecting for the transgene free plants with desired modification in the first generation only.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835450&req=5

Figure 5: The generation of transgene free genetically edited (GE) crops. The transgene free homozygous mutants with desired genetic modifications at the targeted loci and without RGEN transgene construct could be selected by selfing of GE0 generation plants and after segregation of the transgene in the next GE1 generation. The GE plants could be selected by PCR/RE genotyping and DNA sequencing of clones and negatively selecting for the transgene free plants with desired modification in the first generation only.
Mentions: Routine targeted mutagenesis in plants by the CRISPR/Cas9 system will definitely help open up new dimensions in plant biology research. The conventional random mutagenesis approaches were not able to access every gene for inactivation study because of the random nature of the gene integration. CRISPR-Cas9 technology would be of great help in generating mutants for inaccessible genes, mutate multiple loci and generate large deletions, which can, therefore, accelerate plant breeding without actually introducing a transgene (Figure 4). Although, genetically modified crops could have been a solution for crop improvement, if the controversies concerning the probable environmental and health implications of GM crops could be avoided (Hilbeck et al., 2011). Using the technique of genetic engineering a DNA construct can be directly inserted into one or more chromosomes in a random manner for manipulation of the genome. However, the random gene insertions can have undesirable effects and are not favorable for making large intensive changes, such as in case of adding an entire metabolic pathway into a plant (Lau et al., 2014). The plant breeding techniques have been used regularly to introduce new traits into cultivated varieties using existing natural genetic variation and random mutagenesis. The CRISPR/Cas system introduces precise modifications into a plant genome, which inherits stably and transgene region could be removed simply after the target gene editing to make transgene free plants during crop variety improvement (Mahfouz et al., 2014; Gao and Zhao, 2014b; Kanchiswamy et al., 2015; Xu et al., 2015; Figure 5). The Cas9/sgRNA system, is now one of the new plant breeding techniques (NPBT) along with the ZFNs (Zinc Finger Nucleases) and TALENs (Transcription activator like effector nucleases) like genome editing technologies. The NPBTs are faster than traditional breeding methods and can produce a segregant line that notably lacks the transgenic insert (Podevin et al., 2012; Araki and Ishii, 2015; Schaart et al., 2015; Woo et al., 2015). The plants developed by NPBTs are identical to the classically bred plants and these should be evaluated according to the resulting end product rather than the process of creation (Giddings et al., 2012; Hartung and Schiemann, 2014; Woo et al., 2015). Thus, in such a case CRISPR edited plants might be out of the current GMO regulations, but still this pose new challenges for the regulation and social acceptance of genetically edited (GE) crops (Voytas and Gao, 2014; Araki and Ishii, 2015; Jones, 2015; Kanchiswamy et al., 2015). NPBTs are currently in debate by advisory and regulatory authorities in relation to the GMO legislation to classify GE crop varieties produced using genome editing as non-GM for the social acceptance (Lusser et al., 2012; Voytas and Gao, 2014; Araki and Ishii, 2015).

Bottom Line: The Clustered Regularly Interspaced Short Palindromic Repeats associated Cas9/sgRNA system is a novel targeted genome-editing technique derived from bacterial immune system.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.

View Article: PubMed Central - PubMed

Affiliation: Amity Institute of Biotechnology, Amity University Haryana Gurgaon, India.

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.

No MeSH data available.


Related in: MedlinePlus