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A CRISPR/Cas9 toolkit for multiplex genome editing in plants.

Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ - BMC Plant Biol. (2014)

Bottom Line: To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required.Moreover, the multiple-gene mutations could be inherited by the next generation.We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

View Article: PubMed Central - PubMed

ABSTRACT

Background: To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required.

Results: We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation.

Conclusions: We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

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Related in: MedlinePlus

Validation of pCAMBIA-derived CRISPR/Cas binary vectors inArabidopsis. (A) Physical map of T-DNA of the pCAMBIA-derived vector carrying two-gRNAs targeting two Arabidopsis genes (CHLI1 and CHLI2). The alignment of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (B) Phenotypes of all transgenic seedlings from one screening. The T0 seeds were screened on hygromycin MS plates for 7 days, and all of the hygromycin-resistant seedlings were transferred to a fresh MS plate before photographing. The albino seedlings were numbered.
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Fig7: Validation of pCAMBIA-derived CRISPR/Cas binary vectors inArabidopsis. (A) Physical map of T-DNA of the pCAMBIA-derived vector carrying two-gRNAs targeting two Arabidopsis genes (CHLI1 and CHLI2). The alignment of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (B) Phenotypes of all transgenic seedlings from one screening. The T0 seeds were screened on hygromycin MS plates for 7 days, and all of the hygromycin-resistant seedlings were transferred to a fresh MS plate before photographing. The albino seedlings were numbered.

Mentions: To further validate the toolkit in Arabidopsis, we constructed a pCAMBIA-based vector, pHSE-2gR-CHLI, carrying two gRNAs targeting CHLI1 and CHLI2 (Figure 7), which are the same as the gRNAs employed in a previous study [22]. Simultaneous disruption of CHLI1 and CHLI2 led to an albino phenotype, while chli1 or chli2 single mutants displayed a pale green phenotype [48]. A higher ratio of T1 transgenic Arabidopsis seedlings displayed an albino phenotype (24/36 = 67%) than that reported previously (23/60 = 38%), further demonstrating that the toolkit works well for Arabidopsis. Mutation frequencies could be enhanced further through the use of two or more gRNAs to target two or more target sites of the same gene. With the enhanced mutation efficiencies, somatic mutations could be more efficiently transmitted to the next generation. Thus, the toolkit developed in this study could be used to generate Arabidopsis mutants with high levels of efficiency and specificity.Figure 7


A CRISPR/Cas9 toolkit for multiplex genome editing in plants.

Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ - BMC Plant Biol. (2014)

Validation of pCAMBIA-derived CRISPR/Cas binary vectors inArabidopsis. (A) Physical map of T-DNA of the pCAMBIA-derived vector carrying two-gRNAs targeting two Arabidopsis genes (CHLI1 and CHLI2). The alignment of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (B) Phenotypes of all transgenic seedlings from one screening. The T0 seeds were screened on hygromycin MS plates for 7 days, and all of the hygromycin-resistant seedlings were transferred to a fresh MS plate before photographing. The albino seedlings were numbered.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4262988&req=5

Fig7: Validation of pCAMBIA-derived CRISPR/Cas binary vectors inArabidopsis. (A) Physical map of T-DNA of the pCAMBIA-derived vector carrying two-gRNAs targeting two Arabidopsis genes (CHLI1 and CHLI2). The alignment of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (B) Phenotypes of all transgenic seedlings from one screening. The T0 seeds were screened on hygromycin MS plates for 7 days, and all of the hygromycin-resistant seedlings were transferred to a fresh MS plate before photographing. The albino seedlings were numbered.
Mentions: To further validate the toolkit in Arabidopsis, we constructed a pCAMBIA-based vector, pHSE-2gR-CHLI, carrying two gRNAs targeting CHLI1 and CHLI2 (Figure 7), which are the same as the gRNAs employed in a previous study [22]. Simultaneous disruption of CHLI1 and CHLI2 led to an albino phenotype, while chli1 or chli2 single mutants displayed a pale green phenotype [48]. A higher ratio of T1 transgenic Arabidopsis seedlings displayed an albino phenotype (24/36 = 67%) than that reported previously (23/60 = 38%), further demonstrating that the toolkit works well for Arabidopsis. Mutation frequencies could be enhanced further through the use of two or more gRNAs to target two or more target sites of the same gene. With the enhanced mutation efficiencies, somatic mutations could be more efficiently transmitted to the next generation. Thus, the toolkit developed in this study could be used to generate Arabidopsis mutants with high levels of efficiency and specificity.Figure 7

Bottom Line: To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required.Moreover, the multiple-gene mutations could be inherited by the next generation.We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

View Article: PubMed Central - PubMed

ABSTRACT

Background: To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required.

Results: We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation.

Conclusions: We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

Show MeSH
Related in: MedlinePlus