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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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Validation of the toolkit by targeted mutation of a maize gene. (A) Sequence of a region of maize ZmHKT1 with two target sites indicated. (B) Physical map of T-DNA carrying two-gRNA expression cassettes. The alignment of target of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (C) Mutation analysis of 20 T0 transgenic lines by XcmI or SphI digestion of PCR fragments. The lines used for sequencing analysis are indicated with boxes. (D) Alignment of sequences of mutated alleles identified from cloned PCR fragments from two representative T0 transgenic lines. Highlighting denotes the degree of homology of the aligned fragments, and only aligned regions of interest are displayed. The number of indels of the same type is indicated.
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Fig4: Validation of the toolkit by targeted mutation of a maize gene. (A) Sequence of a region of maize ZmHKT1 with two target sites indicated. (B) Physical map of T-DNA carrying two-gRNA expression cassettes. The alignment of target of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (C) Mutation analysis of 20 T0 transgenic lines by XcmI or SphI digestion of PCR fragments. The lines used for sequencing analysis are indicated with boxes. (D) Alignment of sequences of mutated alleles identified from cloned PCR fragments from two representative T0 transgenic lines. Highlighting denotes the degree of homology of the aligned fragments, and only aligned regions of interest are displayed. The number of indels of the same type is indicated.

Mentions: To test the targeted mutation efficiency of the toolkit in monocots, we generated a pCAMBIA-derived CRISPR/Cas9 binary vector with two gRNA expression cassettes targeting the two adjacent sites of the same maize gene, ZmHKT1 (Figure 4A, B). We analyzed 20 T0 transgenic lines by restriction enzyme digestion of a PCR fragment spanning the two target sites, finding that more than 60% of the transgenic lines had a mutation efficiency of approximately 100% for both target sites (Figure 4C). We cloned and sequenced the PCR fragments from two lines with a mutation efficiency of approximately 100%, finding that sequences between the two target sites were deleted, as shown in Figure 4D. These results indicate that the toolkit can be used for high efficiency targeted mutation in maize and possibly other crops.Figure 4


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 the toolkit by targeted mutation of a maize gene. (A) Sequence of a region of maize ZmHKT1 with two target sites indicated. (B) Physical map of T-DNA carrying two-gRNA expression cassettes. The alignment of target of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (C) Mutation analysis of 20 T0 transgenic lines by XcmI or SphI digestion of PCR fragments. The lines used for sequencing analysis are indicated with boxes. (D) Alignment of sequences of mutated alleles identified from cloned PCR fragments from two representative T0 transgenic lines. Highlighting denotes the degree of homology of the aligned fragments, and only aligned regions of interest are displayed. The number of indels of the same type is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Validation of the toolkit by targeted mutation of a maize gene. (A) Sequence of a region of maize ZmHKT1 with two target sites indicated. (B) Physical map of T-DNA carrying two-gRNA expression cassettes. The alignment of target of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (C) Mutation analysis of 20 T0 transgenic lines by XcmI or SphI digestion of PCR fragments. The lines used for sequencing analysis are indicated with boxes. (D) Alignment of sequences of mutated alleles identified from cloned PCR fragments from two representative T0 transgenic lines. Highlighting denotes the degree of homology of the aligned fragments, and only aligned regions of interest are displayed. The number of indels of the same type is indicated.
Mentions: To test the targeted mutation efficiency of the toolkit in monocots, we generated a pCAMBIA-derived CRISPR/Cas9 binary vector with two gRNA expression cassettes targeting the two adjacent sites of the same maize gene, ZmHKT1 (Figure 4A, B). We analyzed 20 T0 transgenic lines by restriction enzyme digestion of a PCR fragment spanning the two target sites, finding that more than 60% of the transgenic lines had a mutation efficiency of approximately 100% for both target sites (Figure 4C). We cloned and sequenced the PCR fragments from two lines with a mutation efficiency of approximately 100%, finding that sequences between the two target sites were deleted, as shown in Figure 4D. These results indicate that the toolkit can be used for high efficiency targeted mutation in maize and possibly other crops.Figure 4

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