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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.

Show MeSH
Validation of maize codon-optimized Cas9 and three Pol-III promoters driving gRNA expression in maize protoplasts. (A) Sequence of the target site from the ZmHKT1 locus. The PAM, the putative cleavage site (red arrowhead), and the XcmI site (boxed) are indicated. (B,C) Mutation analysis by XcmI digestion of PCR fragments. GFP, 201, 301, 401 (B): PCR fragments amplified from the genomic DNA of maize protoplasts transfected with pUC-GFP (control), pBUN201-ZT1, pBUN301-ZT1, and pBUN401-ZT1, respectively. The three CRISPR/Cas9 vectors have the same gRNA but different Cas9: hCas9-1/2, two types of human-codon-optimized Cas9; zCas9, Zea mays codon-optimized Cas9. GFP, 401, 411, 421 (C): PCR fragments from the pUC-GFP, pBUN401-ZT1, pBUN411-ZT1, and pBUN421-ZT1 transfections, respectively; the three CRISPR/Cas9 vectors have the same zCas9 and gRNA, but the gRNA is driven by three different Pol-III promoters. − and + indicate whether the PCR fragments were digested with XcmI. Mutation efficiency (% indel) calculated based on the percent ratios of residual undigested PCR fragments (+ lanes: 569 bp) to total PCR products (− lanes); the WT indel values should be treated as the background level. (D,E) Alignment of sequences of mutated alleles identified from cloned PCR fragments resistant to XcmI digestion. The mutated alleles include deletions (D) and insertions (E). Dots, deleted bases. Highlighting denotes the degree of homology of the aligned fragments, and only aligned regions of interest are shown. The type of indel and the number of indels of the same type are indicated.
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Fig3: Validation of maize codon-optimized Cas9 and three Pol-III promoters driving gRNA expression in maize protoplasts. (A) Sequence of the target site from the ZmHKT1 locus. The PAM, the putative cleavage site (red arrowhead), and the XcmI site (boxed) are indicated. (B,C) Mutation analysis by XcmI digestion of PCR fragments. GFP, 201, 301, 401 (B): PCR fragments amplified from the genomic DNA of maize protoplasts transfected with pUC-GFP (control), pBUN201-ZT1, pBUN301-ZT1, and pBUN401-ZT1, respectively. The three CRISPR/Cas9 vectors have the same gRNA but different Cas9: hCas9-1/2, two types of human-codon-optimized Cas9; zCas9, Zea mays codon-optimized Cas9. GFP, 401, 411, 421 (C): PCR fragments from the pUC-GFP, pBUN401-ZT1, pBUN411-ZT1, and pBUN421-ZT1 transfections, respectively; the three CRISPR/Cas9 vectors have the same zCas9 and gRNA, but the gRNA is driven by three different Pol-III promoters. − and + indicate whether the PCR fragments were digested with XcmI. Mutation efficiency (% indel) calculated based on the percent ratios of residual undigested PCR fragments (+ lanes: 569 bp) to total PCR products (− lanes); the WT indel values should be treated as the background level. (D,E) Alignment of sequences of mutated alleles identified from cloned PCR fragments resistant to XcmI digestion. The mutated alleles include deletions (D) and insertions (E). Dots, deleted bases. Highlighting denotes the degree of homology of the aligned fragments, and only aligned regions of interest are shown. The type of indel and the number of indels of the same type are indicated.

Mentions: For the target site ZT1, the mutated alleles were examined via XcmI digestion of the PCR fragments surrounding the putative cleavage site (Figure 3A). XcmI analysis indicated that maize codon-optimized Cas9 performed considerably better than the two human codon-optimized Cas9 genes (Figure 3B). The TaU3 promoter appeared to perform slightly better than the OsU3 promoter, and the OsU3 promoter performed much better than the AtU6-26 promoter (Figure 3C).Figure 3


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 maize codon-optimized Cas9 and three Pol-III promoters driving gRNA expression in maize protoplasts. (A) Sequence of the target site from the ZmHKT1 locus. The PAM, the putative cleavage site (red arrowhead), and the XcmI site (boxed) are indicated. (B,C) Mutation analysis by XcmI digestion of PCR fragments. GFP, 201, 301, 401 (B): PCR fragments amplified from the genomic DNA of maize protoplasts transfected with pUC-GFP (control), pBUN201-ZT1, pBUN301-ZT1, and pBUN401-ZT1, respectively. The three CRISPR/Cas9 vectors have the same gRNA but different Cas9: hCas9-1/2, two types of human-codon-optimized Cas9; zCas9, Zea mays codon-optimized Cas9. GFP, 401, 411, 421 (C): PCR fragments from the pUC-GFP, pBUN401-ZT1, pBUN411-ZT1, and pBUN421-ZT1 transfections, respectively; the three CRISPR/Cas9 vectors have the same zCas9 and gRNA, but the gRNA is driven by three different Pol-III promoters. − and + indicate whether the PCR fragments were digested with XcmI. Mutation efficiency (% indel) calculated based on the percent ratios of residual undigested PCR fragments (+ lanes: 569 bp) to total PCR products (− lanes); the WT indel values should be treated as the background level. (D,E) Alignment of sequences of mutated alleles identified from cloned PCR fragments resistant to XcmI digestion. The mutated alleles include deletions (D) and insertions (E). Dots, deleted bases. Highlighting denotes the degree of homology of the aligned fragments, and only aligned regions of interest are shown. The type of indel and the number of indels of the same type are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: Validation of maize codon-optimized Cas9 and three Pol-III promoters driving gRNA expression in maize protoplasts. (A) Sequence of the target site from the ZmHKT1 locus. The PAM, the putative cleavage site (red arrowhead), and the XcmI site (boxed) are indicated. (B,C) Mutation analysis by XcmI digestion of PCR fragments. GFP, 201, 301, 401 (B): PCR fragments amplified from the genomic DNA of maize protoplasts transfected with pUC-GFP (control), pBUN201-ZT1, pBUN301-ZT1, and pBUN401-ZT1, respectively. The three CRISPR/Cas9 vectors have the same gRNA but different Cas9: hCas9-1/2, two types of human-codon-optimized Cas9; zCas9, Zea mays codon-optimized Cas9. GFP, 401, 411, 421 (C): PCR fragments from the pUC-GFP, pBUN401-ZT1, pBUN411-ZT1, and pBUN421-ZT1 transfections, respectively; the three CRISPR/Cas9 vectors have the same zCas9 and gRNA, but the gRNA is driven by three different Pol-III promoters. − and + indicate whether the PCR fragments were digested with XcmI. Mutation efficiency (% indel) calculated based on the percent ratios of residual undigested PCR fragments (+ lanes: 569 bp) to total PCR products (− lanes); the WT indel values should be treated as the background level. (D,E) Alignment of sequences of mutated alleles identified from cloned PCR fragments resistant to XcmI digestion. The mutated alleles include deletions (D) and insertions (E). Dots, deleted bases. Highlighting denotes the degree of homology of the aligned fragments, and only aligned regions of interest are shown. The type of indel and the number of indels of the same type are indicated.
Mentions: For the target site ZT1, the mutated alleles were examined via XcmI digestion of the PCR fragments surrounding the putative cleavage site (Figure 3A). XcmI analysis indicated that maize codon-optimized Cas9 performed considerably better than the two human codon-optimized Cas9 genes (Figure 3B). The TaU3 promoter appeared to perform slightly better than the OsU3 promoter, and the OsU3 promoter performed much better than the AtU6-26 promoter (Figure 3C).Figure 3

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