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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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Premade gRNA modules used for the assembly of two to four gRNA expression cassettes. (A) gRNA-expressing modules for both dicots and monocots. U6-29p, U6-26p, and U6-1p are three Arabidopsis U6 gene promoters; U6-29t, U6-26t, and U6-1t, corresponding Arabidopsis U6 gene terminators with downstream sequences; OsU3p and TaU3p, rice and wheat U3 promoters, respectively; OsU3t and TaU3t, rice and wheat U3 terminators with downstream sequences, respectively; gRNA-Sc, gRNA scaffold; DT1/2/3/4, dicot target-1/2/3/4; MT1/2/3/4, monocot target-1/2/3/4. The vector pCBC is the cloning vector into which the gRNA modules were inserted separately. (B) Examples of the assembly of two-gRNA expression cassettes for dicots and monocots using the gRNA modules. Note: Each PCR fragment is flanked by two BsaI sites (not shown).
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Fig2: Premade gRNA modules used for the assembly of two to four gRNA expression cassettes. (A) gRNA-expressing modules for both dicots and monocots. U6-29p, U6-26p, and U6-1p are three Arabidopsis U6 gene promoters; U6-29t, U6-26t, and U6-1t, corresponding Arabidopsis U6 gene terminators with downstream sequences; OsU3p and TaU3p, rice and wheat U3 promoters, respectively; OsU3t and TaU3t, rice and wheat U3 terminators with downstream sequences, respectively; gRNA-Sc, gRNA scaffold; DT1/2/3/4, dicot target-1/2/3/4; MT1/2/3/4, monocot target-1/2/3/4. The vector pCBC is the cloning vector into which the gRNA modules were inserted separately. (B) Examples of the assembly of two-gRNA expression cassettes for dicots and monocots using the gRNA modules. Note: Each PCR fragment is flanked by two BsaI sites (not shown).

Mentions: In order to integrate multiple gRNAs into a single binary vector for multiplex genome editing, we constructed six gRNA module vectors, including three designed for dicots and three designed for monocots (FigureĀ 2). Using these gRNA module vectors, two to more gRNA expression cassettes could easily be assembled using the Golden Gate cloning method [44,45] or the Gibson Assembly method [46]. By employing more suitable Pol III promoters, additional gRNA modules can be constructed for the assembly of more gRNA expression cassettes. Therefore, the gRNA module vector set is extensible and can easily be updated.Figure 2


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)

Premade gRNA modules used for the assembly of two to four gRNA expression cassettes. (A) gRNA-expressing modules for both dicots and monocots. U6-29p, U6-26p, and U6-1p are three Arabidopsis U6 gene promoters; U6-29t, U6-26t, and U6-1t, corresponding Arabidopsis U6 gene terminators with downstream sequences; OsU3p and TaU3p, rice and wheat U3 promoters, respectively; OsU3t and TaU3t, rice and wheat U3 terminators with downstream sequences, respectively; gRNA-Sc, gRNA scaffold; DT1/2/3/4, dicot target-1/2/3/4; MT1/2/3/4, monocot target-1/2/3/4. The vector pCBC is the cloning vector into which the gRNA modules were inserted separately. (B) Examples of the assembly of two-gRNA expression cassettes for dicots and monocots using the gRNA modules. Note: Each PCR fragment is flanked by two BsaI sites (not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Premade gRNA modules used for the assembly of two to four gRNA expression cassettes. (A) gRNA-expressing modules for both dicots and monocots. U6-29p, U6-26p, and U6-1p are three Arabidopsis U6 gene promoters; U6-29t, U6-26t, and U6-1t, corresponding Arabidopsis U6 gene terminators with downstream sequences; OsU3p and TaU3p, rice and wheat U3 promoters, respectively; OsU3t and TaU3t, rice and wheat U3 terminators with downstream sequences, respectively; gRNA-Sc, gRNA scaffold; DT1/2/3/4, dicot target-1/2/3/4; MT1/2/3/4, monocot target-1/2/3/4. The vector pCBC is the cloning vector into which the gRNA modules were inserted separately. (B) Examples of the assembly of two-gRNA expression cassettes for dicots and monocots using the gRNA modules. Note: Each PCR fragment is flanked by two BsaI sites (not shown).
Mentions: In order to integrate multiple gRNAs into a single binary vector for multiplex genome editing, we constructed six gRNA module vectors, including three designed for dicots and three designed for monocots (FigureĀ 2). Using these gRNA module vectors, two to more gRNA expression cassettes could easily be assembled using the Golden Gate cloning method [44,45] or the Gibson Assembly method [46]. By employing more suitable Pol III promoters, additional gRNA modules can be constructed for the assembly of more gRNA expression cassettes. Therefore, the gRNA module vector set is extensible and can easily be updated.Figure 2

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