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Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice.

Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP - Nucleic Acids Res. (2013)

Bottom Line: Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum.Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco.Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Nebraska, Lincoln, NE 68588, USA, Deparment of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA and Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE 68588, USA.

ABSTRACT
The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5' coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.

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DNA sequences of initially non-functional GFP genes following target site cleavage by Cas9/sgRNA and mutagenesis by NHEJ DNA repair in transgenic Arabidopsis and tobacco leaf cells expressing Cas9 and sgRNA genes. The DNA sequence of the starting non-functional mutant GFP gene is provided above each set of Cas9/sgRNA-mutagenized gene sequences derived from Arabidopsis (top set) and tobacco (bottom set). The 20 nt target sequence for the Cas9/sgRNA complex is in blue, the PAM site in red, and the ApaLI recognition site is underlined in blue. For the Cas9/sgRNA-mutagenized DNA sequences, deleted nucleotides are depicted as red dots and inserted nucleotides are shown in green. The net length of insertions and/or deleletions (In/Del) and the frequency with which each DNA sequence pattern was observed (Freq.) are presented in the columns to the right. One sequence in the Arabidopsis set and one sequence in the tobacco set arose from sequential deletions and insertions (first and second lines of each DNA sequence set, respectively).
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gkt780-F5: DNA sequences of initially non-functional GFP genes following target site cleavage by Cas9/sgRNA and mutagenesis by NHEJ DNA repair in transgenic Arabidopsis and tobacco leaf cells expressing Cas9 and sgRNA genes. The DNA sequence of the starting non-functional mutant GFP gene is provided above each set of Cas9/sgRNA-mutagenized gene sequences derived from Arabidopsis (top set) and tobacco (bottom set). The 20 nt target sequence for the Cas9/sgRNA complex is in blue, the PAM site in red, and the ApaLI recognition site is underlined in blue. For the Cas9/sgRNA-mutagenized DNA sequences, deleted nucleotides are depicted as red dots and inserted nucleotides are shown in green. The net length of insertions and/or deleletions (In/Del) and the frequency with which each DNA sequence pattern was observed (Freq.) are presented in the columns to the right. One sequence in the Arabidopsis set and one sequence in the tobacco set arose from sequential deletions and insertions (first and second lines of each DNA sequence set, respectively).

Mentions: To establish that the Cas9/sgRNA system causes mutations at the target site in the non-functional GFP genes in Arabidopsis and tobacco leaf cells is responsible for the conversion of the non-functional GFP gene into a functional GFP gene, DNA was extracted from leaf regions displaying fluorescence and subjected to PCR amplification using primers annealing to sites 125-bp upstream and 125-bp downstream of the Cas9-sgRNA target site. By digesting the extracted DNA with the restriction enzyme, ApaLI before amplification, advantage was taken of the presence of a ApaLI restriction site at the targeted Cas9-sgRNA cut site of the nonfunctional GFP to prevent the amplification of DNA from the non-functional GFP gene but allow amplification of mutagenized GFP gene segments in which the ApaLI cut site had been destroyed by nucleotide insertions or deletions created during DNA repair by the NHEJ repair system. DNA sequencing of cloned PCR products showed that approximately one-half of the cloned DNA sequences were from non-mutagenized non-functional GFP genes that apparently escaped ApaLI digestion (Figure 5). Nonetheless, the procedure for enrichment of mutagenized GFP gene DNA segments was successful in allowing recovery of clones of PCR-amplified DNA bearing six different DNA sequence patterns from Arabidopsis leaves and eight different DNA sequence patterns from tobacco leaves (Figure 5). As is common with NHEJ DNA repair, most mutations resulted from small deletions ranging from 1 to 17 nt or insertion of 1 to 3 nt. In two cases, there were apparent combinations of small deletions and insertions.Figure 5.


Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice.

Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP - Nucleic Acids Res. (2013)

DNA sequences of initially non-functional GFP genes following target site cleavage by Cas9/sgRNA and mutagenesis by NHEJ DNA repair in transgenic Arabidopsis and tobacco leaf cells expressing Cas9 and sgRNA genes. The DNA sequence of the starting non-functional mutant GFP gene is provided above each set of Cas9/sgRNA-mutagenized gene sequences derived from Arabidopsis (top set) and tobacco (bottom set). The 20 nt target sequence for the Cas9/sgRNA complex is in blue, the PAM site in red, and the ApaLI recognition site is underlined in blue. For the Cas9/sgRNA-mutagenized DNA sequences, deleted nucleotides are depicted as red dots and inserted nucleotides are shown in green. The net length of insertions and/or deleletions (In/Del) and the frequency with which each DNA sequence pattern was observed (Freq.) are presented in the columns to the right. One sequence in the Arabidopsis set and one sequence in the tobacco set arose from sequential deletions and insertions (first and second lines of each DNA sequence set, respectively).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814374&req=5

gkt780-F5: DNA sequences of initially non-functional GFP genes following target site cleavage by Cas9/sgRNA and mutagenesis by NHEJ DNA repair in transgenic Arabidopsis and tobacco leaf cells expressing Cas9 and sgRNA genes. The DNA sequence of the starting non-functional mutant GFP gene is provided above each set of Cas9/sgRNA-mutagenized gene sequences derived from Arabidopsis (top set) and tobacco (bottom set). The 20 nt target sequence for the Cas9/sgRNA complex is in blue, the PAM site in red, and the ApaLI recognition site is underlined in blue. For the Cas9/sgRNA-mutagenized DNA sequences, deleted nucleotides are depicted as red dots and inserted nucleotides are shown in green. The net length of insertions and/or deleletions (In/Del) and the frequency with which each DNA sequence pattern was observed (Freq.) are presented in the columns to the right. One sequence in the Arabidopsis set and one sequence in the tobacco set arose from sequential deletions and insertions (first and second lines of each DNA sequence set, respectively).
Mentions: To establish that the Cas9/sgRNA system causes mutations at the target site in the non-functional GFP genes in Arabidopsis and tobacco leaf cells is responsible for the conversion of the non-functional GFP gene into a functional GFP gene, DNA was extracted from leaf regions displaying fluorescence and subjected to PCR amplification using primers annealing to sites 125-bp upstream and 125-bp downstream of the Cas9-sgRNA target site. By digesting the extracted DNA with the restriction enzyme, ApaLI before amplification, advantage was taken of the presence of a ApaLI restriction site at the targeted Cas9-sgRNA cut site of the nonfunctional GFP to prevent the amplification of DNA from the non-functional GFP gene but allow amplification of mutagenized GFP gene segments in which the ApaLI cut site had been destroyed by nucleotide insertions or deletions created during DNA repair by the NHEJ repair system. DNA sequencing of cloned PCR products showed that approximately one-half of the cloned DNA sequences were from non-mutagenized non-functional GFP genes that apparently escaped ApaLI digestion (Figure 5). Nonetheless, the procedure for enrichment of mutagenized GFP gene DNA segments was successful in allowing recovery of clones of PCR-amplified DNA bearing six different DNA sequence patterns from Arabidopsis leaves and eight different DNA sequence patterns from tobacco leaves (Figure 5). As is common with NHEJ DNA repair, most mutations resulted from small deletions ranging from 1 to 17 nt or insertion of 1 to 3 nt. In two cases, there were apparent combinations of small deletions and insertions.Figure 5.

Bottom Line: Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum.Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco.Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Nebraska, Lincoln, NE 68588, USA, Deparment of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA and Department of Agronomy and Horticulture, University of Nebraska, Lincoln, NE 68588, USA.

ABSTRACT
The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5' coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.

Show MeSH
Related in: MedlinePlus