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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 basic strategy of Cas9/sgRNA system. The Cas9 is a RNA guided endonuclease consists of two nuclease domains namely HNH and RuvC. The target specificity of Cas9 depends upon the guide sequence (20 nt) short guide RNA (sgRNA). The target sites must lay immediately 5′of a PAM (Protospacer Adjacent Motif) sequence of the form N20-NGG (or N20-NAG). The Cas9 nuclease induces double stranded breaks (DSB) at the target site which can be repaired either by Non- Homologous End Joining method or Homologous Recombination by cellular system which results in gene disruption by indels or gene addition/correction, respectively.
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Figure 1: The basic strategy of Cas9/sgRNA system. The Cas9 is a RNA guided endonuclease consists of two nuclease domains namely HNH and RuvC. The target specificity of Cas9 depends upon the guide sequence (20 nt) short guide RNA (sgRNA). The target sites must lay immediately 5′of a PAM (Protospacer Adjacent Motif) sequence of the form N20-NGG (or N20-NAG). The Cas9 nuclease induces double stranded breaks (DSB) at the target site which can be repaired either by Non- Homologous End Joining method or Homologous Recombination by cellular system which results in gene disruption by indels or gene addition/correction, respectively.

Mentions: Clustered regularly interspaced short palindromic repeat/Cas system was discovered in bacteria as an adaptive immune system which helps the bacteria in protecting itself against invading foreign DNA, such as that of a bacteriophage. This system comprises of CRISPR loci in the genome and a Cas9 protein. CRISPR, i.e., Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) – is a genomic locus of tandem direct repeat sequences and protospacers, the spaces in between repeat sequences, both of which are derived from the invading elements (Kim and Kim, 2014). The CRISPR loci contains a combination of Cas9 genes; sequences for non-coding RNA elements called CRISPR RNA (crRNA) and sequences for small trans-encoded CRISPR RNA, i.e., trans-activating crRNA (tracrRNA). The two RNA sequences crRNA and tracrRNA forms a complex known as guide RNA, which determines the specificity of the cleavage of the target sequence in the nucleic acid along with the Protosapcer Adjacent Motif (PAM), a 5′-NGG sequence (Barrangou, 2013; Jinek et al., 2013). The cleavage of the double stranded target DNA occurs within the limits of protospacer region. The Cas9 protein is an endonuclease associated with CRISPR loci, which is responsible for the double-stranded breaks (DSBs) at the site, when targeted by a guide RNA (Cong et al., 2013; Mali et al., 2013). This revealing of molecular mechanism of the CRISPR/Cas system in 2012 opened up its vast area of applications as a promising component of genome editing termed as RNA-guided engineered nucleases (RGENs), which were used as sequence specific nucleases for precise genetic modifications (Doudna and Charpentier, 2014; Fichtner et al., 2014; Liang et al., 2015). RGENs are developed as programmable nucleases composed of two components, which must be expressed in cells to perform genome editing; the Cas9 nuclease and an engineered single guide RNA (sgRNA). The sgRNA has 20 nucleotides at the 5′ end that directs Cas9 to the complementary target site. Any DNA sequence of the form N20-NGG can be targeted by altering the first 20 nucleotides of the gRNA for novel genome editing applications (Sander and Joung, 2014) (Figure 1).


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 basic strategy of Cas9/sgRNA system. The Cas9 is a RNA guided endonuclease consists of two nuclease domains namely HNH and RuvC. The target specificity of Cas9 depends upon the guide sequence (20 nt) short guide RNA (sgRNA). The target sites must lay immediately 5′of a PAM (Protospacer Adjacent Motif) sequence of the form N20-NGG (or N20-NAG). The Cas9 nuclease induces double stranded breaks (DSB) at the target site which can be repaired either by Non- Homologous End Joining method or Homologous Recombination by cellular system which results in gene disruption by indels or gene addition/correction, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: The basic strategy of Cas9/sgRNA system. The Cas9 is a RNA guided endonuclease consists of two nuclease domains namely HNH and RuvC. The target specificity of Cas9 depends upon the guide sequence (20 nt) short guide RNA (sgRNA). The target sites must lay immediately 5′of a PAM (Protospacer Adjacent Motif) sequence of the form N20-NGG (or N20-NAG). The Cas9 nuclease induces double stranded breaks (DSB) at the target site which can be repaired either by Non- Homologous End Joining method or Homologous Recombination by cellular system which results in gene disruption by indels or gene addition/correction, respectively.
Mentions: Clustered regularly interspaced short palindromic repeat/Cas system was discovered in bacteria as an adaptive immune system which helps the bacteria in protecting itself against invading foreign DNA, such as that of a bacteriophage. This system comprises of CRISPR loci in the genome and a Cas9 protein. CRISPR, i.e., Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) – is a genomic locus of tandem direct repeat sequences and protospacers, the spaces in between repeat sequences, both of which are derived from the invading elements (Kim and Kim, 2014). The CRISPR loci contains a combination of Cas9 genes; sequences for non-coding RNA elements called CRISPR RNA (crRNA) and sequences for small trans-encoded CRISPR RNA, i.e., trans-activating crRNA (tracrRNA). The two RNA sequences crRNA and tracrRNA forms a complex known as guide RNA, which determines the specificity of the cleavage of the target sequence in the nucleic acid along with the Protosapcer Adjacent Motif (PAM), a 5′-NGG sequence (Barrangou, 2013; Jinek et al., 2013). The cleavage of the double stranded target DNA occurs within the limits of protospacer region. The Cas9 protein is an endonuclease associated with CRISPR loci, which is responsible for the double-stranded breaks (DSBs) at the site, when targeted by a guide RNA (Cong et al., 2013; Mali et al., 2013). This revealing of molecular mechanism of the CRISPR/Cas system in 2012 opened up its vast area of applications as a promising component of genome editing termed as RNA-guided engineered nucleases (RGENs), which were used as sequence specific nucleases for precise genetic modifications (Doudna and Charpentier, 2014; Fichtner et al., 2014; Liang et al., 2015). RGENs are developed as programmable nucleases composed of two components, which must be expressed in cells to perform genome editing; the Cas9 nuclease and an engineered single guide RNA (sgRNA). The sgRNA has 20 nucleotides at the 5′ end that directs Cas9 to the complementary target site. Any DNA sequence of the form N20-NGG can be targeted by altering the first 20 nucleotides of the gRNA for novel genome editing applications (Sander and Joung, 2014) (Figure 1).

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