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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 different variants of Cas9/sgRNA system of genome editing.(A) The Cas9 nickase (Cas9n) with a RuvC or HNH mutation create a nick instead of a DSB at the target site. The dimeric Cas9n can be used for enhances specificity and reduced off target effects. (B) Dimeric RNA-guided FokI nucleases (RFNs) are the fusion of a catalytically inactive dCas9 protein with the FokI nuclease domain. Dimerization of two RFNs used for high genome editing frequencies and reduced off-target mutations. (C) Ribozyme-gRNA-Ribozyme (RGR) is an artificial gene, which generates self-catalyzed desired gRNA after transcribed from any promoter for tissue-specific genome editing. (D) Primary Polycistronic tRNA-gRNA (PTG) is tandemly arrayed tRNA-gRNA units, which is cleaved by the endogenous tRNA-processing system for simultaneously targeting multiple sites.
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Figure 2: The different variants of Cas9/sgRNA system of genome editing.(A) The Cas9 nickase (Cas9n) with a RuvC or HNH mutation create a nick instead of a DSB at the target site. The dimeric Cas9n can be used for enhances specificity and reduced off target effects. (B) Dimeric RNA-guided FokI nucleases (RFNs) are the fusion of a catalytically inactive dCas9 protein with the FokI nuclease domain. Dimerization of two RFNs used for high genome editing frequencies and reduced off-target mutations. (C) Ribozyme-gRNA-Ribozyme (RGR) is an artificial gene, which generates self-catalyzed desired gRNA after transcribed from any promoter for tissue-specific genome editing. (D) Primary Polycistronic tRNA-gRNA (PTG) is tandemly arrayed tRNA-gRNA units, which is cleaved by the endogenous tRNA-processing system for simultaneously targeting multiple sites.

Mentions: A Cas9 nickase (Cas9n) was first developed by Cong et al. (2013) through a mutation in native Cas9 (D10A, aspartate to alanine substitution). The Cas9 nickase with a RuvC or HNH mutation has the ability to create a nick, instead of a DSB at the target site. The individual nicks in the genome can be typically repaired with high fidelity homology-directed repair (HDR). The Cas9n has been used into a paired nickase system with two different gRNA to extend the number of specifically recognized bases for target cleavage, which has improved the specificity and helped mitigate the off-target phenomena (Cong et al., 2013; Mali et al., 2013; Ran et al., 2013; Fauser et al., 2014). The paired-nicking strategy has high-efficiency of HDR with reduced off-target cleavages by 50- to 1,500-fold in human cells, without sacrificing on-target cleavage efficiency (Ran et al., 2013) (Figure 2A).


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 different variants of Cas9/sgRNA system of genome editing.(A) The Cas9 nickase (Cas9n) with a RuvC or HNH mutation create a nick instead of a DSB at the target site. The dimeric Cas9n can be used for enhances specificity and reduced off target effects. (B) Dimeric RNA-guided FokI nucleases (RFNs) are the fusion of a catalytically inactive dCas9 protein with the FokI nuclease domain. Dimerization of two RFNs used for high genome editing frequencies and reduced off-target mutations. (C) Ribozyme-gRNA-Ribozyme (RGR) is an artificial gene, which generates self-catalyzed desired gRNA after transcribed from any promoter for tissue-specific genome editing. (D) Primary Polycistronic tRNA-gRNA (PTG) is tandemly arrayed tRNA-gRNA units, which is cleaved by the endogenous tRNA-processing system for simultaneously targeting multiple sites.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: The different variants of Cas9/sgRNA system of genome editing.(A) The Cas9 nickase (Cas9n) with a RuvC or HNH mutation create a nick instead of a DSB at the target site. The dimeric Cas9n can be used for enhances specificity and reduced off target effects. (B) Dimeric RNA-guided FokI nucleases (RFNs) are the fusion of a catalytically inactive dCas9 protein with the FokI nuclease domain. Dimerization of two RFNs used for high genome editing frequencies and reduced off-target mutations. (C) Ribozyme-gRNA-Ribozyme (RGR) is an artificial gene, which generates self-catalyzed desired gRNA after transcribed from any promoter for tissue-specific genome editing. (D) Primary Polycistronic tRNA-gRNA (PTG) is tandemly arrayed tRNA-gRNA units, which is cleaved by the endogenous tRNA-processing system for simultaneously targeting multiple sites.
Mentions: A Cas9 nickase (Cas9n) was first developed by Cong et al. (2013) through a mutation in native Cas9 (D10A, aspartate to alanine substitution). The Cas9 nickase with a RuvC or HNH mutation has the ability to create a nick, instead of a DSB at the target site. The individual nicks in the genome can be typically repaired with high fidelity homology-directed repair (HDR). The Cas9n has been used into a paired nickase system with two different gRNA to extend the number of specifically recognized bases for target cleavage, which has improved the specificity and helped mitigate the off-target phenomena (Cong et al., 2013; Mali et al., 2013; Ran et al., 2013; Fauser et al., 2014). The paired-nicking strategy has high-efficiency of HDR with reduced off-target cleavages by 50- to 1,500-fold in human cells, without sacrificing on-target cleavage efficiency (Ran et al., 2013) (Figure 2A).

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