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Efficient CRISPR/Cas9-mediated Targeted Mutagenesis in Populus in the First Generation.

Fan D, Liu T, Li C, Jiao B, Li S, Hou Y, Luo K - Sci Rep (2015)

Bottom Line: Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been applied to edit the plant genome in several herbaceous plant species.In this study, we describe the genome editing and targeted gene mutation in a woody species, Populus tomentosa Carr. via the CRISPR/Cas9 system.Our data demonstrate that the Cas9/sgRNA system can be exploited to precisely edit genomic sequence and effectively create knockout mutations in woody plants.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Eco-environments of Three Gorges Reservoir Region, Ministry of Education, Chongqing Key Laboratory of Transgenic Plant and Safety Control, Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing 400715, China.

ABSTRACT
Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been applied to edit the plant genome in several herbaceous plant species. However, it remains unknown whether this system can be used for genome editing in woody plants. In this study, we describe the genome editing and targeted gene mutation in a woody species, Populus tomentosa Carr. via the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed to target with distinct poplar genomic sites of the phytoene desaturase gene 8 (PtoPDS) which are followed by the protospacer-adjacent motif (PAM). After Agrobacterium-mediated transformation, obvious albino phenotype was observed in transgenic poplar plants. By analyzing the RNA-guided genome-editing events, 30 out of 59 PCR clones were homozygous mutants, 2 out of 59 were heterozygous mutants and the mutation efficiency at these target sites was estimated to be 51.7%. Our data demonstrate that the Cas9/sgRNA system can be exploited to precisely edit genomic sequence and effectively create knockout mutations in woody plants.

No MeSH data available.


Related in: MedlinePlus

Schematic diagram of assembling Cas9/sgRNA construct and selecting target sites in the PtoPDS gene.(A) Schematic illustrating the four sgRNAs (red lines) targeting the PtoPDS coding sequence. Blue boxes indicate exons; orange lines indicate introns; target sites 2 and 3 are titllingly arranged. F1, F2, R1, R2 indicate binding sites of the primers using for PCR amplification. (B) Schematic view of the method for constructing the expression cassettes of sgRNAs. Left, the backbone of sgRNA that any specific targeting sequence can be inserted between the promoter and the unchanged part of guide-RNA using BsaI. Right, the four promters from Arabidopsis, AtU3b, AtU3d, AtU6-1, AtU6-29 were used to drive the four PtoPDS targeted sgRNAs, respectively. (C) Schematic diagram of the assembling of sgRNAs and Cas9 expression cassettes in a single binary vector for plant stable transformation mediated by Agrobacterium. By the design tails after cutting with BsaI, four sgRNA expression cassettes were ligated into the binary vector sequentially.
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f1: Schematic diagram of assembling Cas9/sgRNA construct and selecting target sites in the PtoPDS gene.(A) Schematic illustrating the four sgRNAs (red lines) targeting the PtoPDS coding sequence. Blue boxes indicate exons; orange lines indicate introns; target sites 2 and 3 are titllingly arranged. F1, F2, R1, R2 indicate binding sites of the primers using for PCR amplification. (B) Schematic view of the method for constructing the expression cassettes of sgRNAs. Left, the backbone of sgRNA that any specific targeting sequence can be inserted between the promoter and the unchanged part of guide-RNA using BsaI. Right, the four promters from Arabidopsis, AtU3b, AtU3d, AtU6-1, AtU6-29 were used to drive the four PtoPDS targeted sgRNAs, respectively. (C) Schematic diagram of the assembling of sgRNAs and Cas9 expression cassettes in a single binary vector for plant stable transformation mediated by Agrobacterium. By the design tails after cutting with BsaI, four sgRNA expression cassettes were ligated into the binary vector sequentially.

Mentions: In order to test whether the CRSPR/Cas9 system could effectively direct gene-specific editing in Populus, we selected the poplar phytoene desaturase gene (PtoPDS) , which is required for chlorophyll biosynthesis and its mutant shows an albino phenotype in other plant species924, as the target of Cas9 endonuclease. Four 20-bp sequences with tandem guanosine nucleotides (PAM) on their 3´-regions in PtoPDS locus were elected as sgRNA complementary sites, including one in 5´ of first exon and three in the second exon (Supplementary Figure S1 and Fig. 1A). By a two-step assemble strategy, the four targeting sequences aiming to PtoPDS were first inserted in the sgRNA expression cassettes (Fig. 1B), and then their cassettes were combined with the Cas9 endonuclease coding sequence in a single plant binary vector, pYLCRIPSR/Cas9P35S-H (Fig. 1C). Using this system and Agrobacterium-mediated transformation, Cas9 and four sgRNAs could simultaneously express in transgenic poplar.


Efficient CRISPR/Cas9-mediated Targeted Mutagenesis in Populus in the First Generation.

Fan D, Liu T, Li C, Jiao B, Li S, Hou Y, Luo K - Sci Rep (2015)

Schematic diagram of assembling Cas9/sgRNA construct and selecting target sites in the PtoPDS gene.(A) Schematic illustrating the four sgRNAs (red lines) targeting the PtoPDS coding sequence. Blue boxes indicate exons; orange lines indicate introns; target sites 2 and 3 are titllingly arranged. F1, F2, R1, R2 indicate binding sites of the primers using for PCR amplification. (B) Schematic view of the method for constructing the expression cassettes of sgRNAs. Left, the backbone of sgRNA that any specific targeting sequence can be inserted between the promoter and the unchanged part of guide-RNA using BsaI. Right, the four promters from Arabidopsis, AtU3b, AtU3d, AtU6-1, AtU6-29 were used to drive the four PtoPDS targeted sgRNAs, respectively. (C) Schematic diagram of the assembling of sgRNAs and Cas9 expression cassettes in a single binary vector for plant stable transformation mediated by Agrobacterium. By the design tails after cutting with BsaI, four sgRNA expression cassettes were ligated into the binary vector sequentially.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507398&req=5

f1: Schematic diagram of assembling Cas9/sgRNA construct and selecting target sites in the PtoPDS gene.(A) Schematic illustrating the four sgRNAs (red lines) targeting the PtoPDS coding sequence. Blue boxes indicate exons; orange lines indicate introns; target sites 2 and 3 are titllingly arranged. F1, F2, R1, R2 indicate binding sites of the primers using for PCR amplification. (B) Schematic view of the method for constructing the expression cassettes of sgRNAs. Left, the backbone of sgRNA that any specific targeting sequence can be inserted between the promoter and the unchanged part of guide-RNA using BsaI. Right, the four promters from Arabidopsis, AtU3b, AtU3d, AtU6-1, AtU6-29 were used to drive the four PtoPDS targeted sgRNAs, respectively. (C) Schematic diagram of the assembling of sgRNAs and Cas9 expression cassettes in a single binary vector for plant stable transformation mediated by Agrobacterium. By the design tails after cutting with BsaI, four sgRNA expression cassettes were ligated into the binary vector sequentially.
Mentions: In order to test whether the CRSPR/Cas9 system could effectively direct gene-specific editing in Populus, we selected the poplar phytoene desaturase gene (PtoPDS) , which is required for chlorophyll biosynthesis and its mutant shows an albino phenotype in other plant species924, as the target of Cas9 endonuclease. Four 20-bp sequences with tandem guanosine nucleotides (PAM) on their 3´-regions in PtoPDS locus were elected as sgRNA complementary sites, including one in 5´ of first exon and three in the second exon (Supplementary Figure S1 and Fig. 1A). By a two-step assemble strategy, the four targeting sequences aiming to PtoPDS were first inserted in the sgRNA expression cassettes (Fig. 1B), and then their cassettes were combined with the Cas9 endonuclease coding sequence in a single plant binary vector, pYLCRIPSR/Cas9P35S-H (Fig. 1C). Using this system and Agrobacterium-mediated transformation, Cas9 and four sgRNAs could simultaneously express in transgenic poplar.

Bottom Line: Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been applied to edit the plant genome in several herbaceous plant species.In this study, we describe the genome editing and targeted gene mutation in a woody species, Populus tomentosa Carr. via the CRISPR/Cas9 system.Our data demonstrate that the Cas9/sgRNA system can be exploited to precisely edit genomic sequence and effectively create knockout mutations in woody plants.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Eco-environments of Three Gorges Reservoir Region, Ministry of Education, Chongqing Key Laboratory of Transgenic Plant and Safety Control, Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing 400715, China.

ABSTRACT
Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been applied to edit the plant genome in several herbaceous plant species. However, it remains unknown whether this system can be used for genome editing in woody plants. In this study, we describe the genome editing and targeted gene mutation in a woody species, Populus tomentosa Carr. via the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed to target with distinct poplar genomic sites of the phytoene desaturase gene 8 (PtoPDS) which are followed by the protospacer-adjacent motif (PAM). After Agrobacterium-mediated transformation, obvious albino phenotype was observed in transgenic poplar plants. By analyzing the RNA-guided genome-editing events, 30 out of 59 PCR clones were homozygous mutants, 2 out of 59 were heterozygous mutants and the mutation efficiency at these target sites was estimated to be 51.7%. Our data demonstrate that the Cas9/sgRNA system can be exploited to precisely edit genomic sequence and effectively create knockout mutations in woody plants.

No MeSH data available.


Related in: MedlinePlus