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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 the CRISPR/Cas toolkit inArabidopsis. (A) Physical maps of the T-DNAs of two pGreen-derived CRISPR/Cas9 binary vectors, each carrying two-gRNAs targeting three Arabidopsis genes (TRY, CPC and ETC2). The alignment of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (B) Representative phenotypes of p2gR-TRI-A T1 transgenic lines. S, strong phenotypes similar to that of try cpc etc2 triple mutant, with highly clustered trichomes on leaf blades and petioles; M, moderate phenotypes with parts of leaf blades or a partial leaf blade displaying the phenotypes of the try cpc double mutant or the triple mutant; W, plants with weak or no mutant phenotypes. The total number of T1 transgenic plants, the number of T1 transgenic plants displaying strong, moderate, and weak phenotypes, and the percentage (in parentheses) of the total number are shown. The T0 seeds were screened on hygromycin MS plates for 13 days and grown in soil for 10 days before photographing. (C) Magnified image of a detached leaf displaying highly clustered trichomes on petioles, which is similar to the phenotype of the try cpc etc2 triple mutant. (D) Sequencing analysis of target gene mutations of a representative p2gR-TRI-A line. Dots, deleted bases. Highlighting denotes the degree of homology of the aligned fragments. The type of indel and the number of indels of the same type are indicated.
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Fig5: Validation of the CRISPR/Cas toolkit inArabidopsis. (A) Physical maps of the T-DNAs of two pGreen-derived CRISPR/Cas9 binary vectors, each carrying two-gRNAs targeting three Arabidopsis genes (TRY, CPC and ETC2). The alignment of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (B) Representative phenotypes of p2gR-TRI-A T1 transgenic lines. S, strong phenotypes similar to that of try cpc etc2 triple mutant, with highly clustered trichomes on leaf blades and petioles; M, moderate phenotypes with parts of leaf blades or a partial leaf blade displaying the phenotypes of the try cpc double mutant or the triple mutant; W, plants with weak or no mutant phenotypes. The total number of T1 transgenic plants, the number of T1 transgenic plants displaying strong, moderate, and weak phenotypes, and the percentage (in parentheses) of the total number are shown. The T0 seeds were screened on hygromycin MS plates for 13 days and grown in soil for 10 days before photographing. (C) Magnified image of a detached leaf displaying highly clustered trichomes on petioles, which is similar to the phenotype of the try cpc etc2 triple mutant. (D) Sequencing analysis of target gene mutations of a representative p2gR-TRI-A line. Dots, deleted bases. Highlighting denotes the degree of homology of the aligned fragments. The type of indel and the number of indels of the same type are indicated.

Mentions: Two vectors, p2gR-TRI-A and p2gR-TRI-B (Figure 5A), each carrying two gRNAs targeting three genes related to trichome development, were used to transform Arabidopsis. Both vectors contain the same gRNA (T2-ETC2), which targets ETC2 and possibly CPC, a much less favorable target (Figure 5A). The vectors also contain different gRNAs (T1A-TC or T1B-TC). The 18-bp target sequence in T1A-TC is reversely complementary to that in T1B-TC. Both T1A-TC and T1B-TC target the same two genes: TRY and CPC (Figure 5A). There is only one mismatch between the 20-nt target of T1A-TC gRNA and TRY or CPC and between that of T1B-TC and TRY, whereas there are two mismatches between that of T1B-TC and CPC (Figure 5A). For p2gR-TRI-A, more than 70% of the T1 transgenic plants displayed highly clustered trichomes (Figure 5B,C), as expected for try cpc double or try cpc etc2 triple mutant plants [47]. For p2gR-TRI-B, less than 10% of the plants displayed the expected phenotypes, which suggests that T1B-TC has a much less favorable performance level than T1A-TC. Sequencing of the mutated alleles from a p2gR-TRI-B T1 transgenic line revealed that although the mutation efficiency of the TRY allele was more than 90%, that of CPC resulting from the same T1B-TC gRNA was only 42% (Additional file 1: Figure S1). By contrast, both CPC and TRY targeted by the same T1A-TC gRNA had similar mutation frequencies (greater than 90%), regardless of the fact that there were different PAMs between the two target sites (Figure 5A, D). These results suggest that the two mismatches might explain the poor performance of T1B-TC, although the two mismatches are located at the 5’-end of the gRNA. Furthermore, when there were three mismatches between the 20-nt target sequence of T2-ETC2 gRNA and the targeted gene CPC (Figure 5A), no mutation was detectable in more than 100 clones from the p2gR-TRI-A transgenic line. By contrast, ETC2 from the same T2-ETC2 gRNA had a mutation efficiency of 72% (Figure 5D). These results indicate that in planta, the CRISPR/Cas9 system has high sequence specificity, and two or more mismatches can greatly reduce the targeting efficiency and off-target effects, especially when a mismatch is near the 3′-end of the 20-nt target of a gRNA.Figure 5


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 CRISPR/Cas toolkit inArabidopsis. (A) Physical maps of the T-DNAs of two pGreen-derived CRISPR/Cas9 binary vectors, each carrying two-gRNAs targeting three Arabidopsis genes (TRY, CPC and ETC2). The alignment of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (B) Representative phenotypes of p2gR-TRI-A T1 transgenic lines. S, strong phenotypes similar to that of try cpc etc2 triple mutant, with highly clustered trichomes on leaf blades and petioles; M, moderate phenotypes with parts of leaf blades or a partial leaf blade displaying the phenotypes of the try cpc double mutant or the triple mutant; W, plants with weak or no mutant phenotypes. The total number of T1 transgenic plants, the number of T1 transgenic plants displaying strong, moderate, and weak phenotypes, and the percentage (in parentheses) of the total number are shown. The T0 seeds were screened on hygromycin MS plates for 13 days and grown in soil for 10 days before photographing. (C) Magnified image of a detached leaf displaying highly clustered trichomes on petioles, which is similar to the phenotype of the try cpc etc2 triple mutant. (D) Sequencing analysis of target gene mutations of a representative p2gR-TRI-A line. Dots, deleted bases. Highlighting denotes the degree of homology of the aligned fragments. The type of indel and the number of indels of the same type are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Fig5: Validation of the CRISPR/Cas toolkit inArabidopsis. (A) Physical maps of the T-DNAs of two pGreen-derived CRISPR/Cas9 binary vectors, each carrying two-gRNAs targeting three Arabidopsis genes (TRY, CPC and ETC2). The alignment of gRNA with its target gene is shown. Only aligned regions of interest are displayed. -rc, reverse complement. (B) Representative phenotypes of p2gR-TRI-A T1 transgenic lines. S, strong phenotypes similar to that of try cpc etc2 triple mutant, with highly clustered trichomes on leaf blades and petioles; M, moderate phenotypes with parts of leaf blades or a partial leaf blade displaying the phenotypes of the try cpc double mutant or the triple mutant; W, plants with weak or no mutant phenotypes. The total number of T1 transgenic plants, the number of T1 transgenic plants displaying strong, moderate, and weak phenotypes, and the percentage (in parentheses) of the total number are shown. The T0 seeds were screened on hygromycin MS plates for 13 days and grown in soil for 10 days before photographing. (C) Magnified image of a detached leaf displaying highly clustered trichomes on petioles, which is similar to the phenotype of the try cpc etc2 triple mutant. (D) Sequencing analysis of target gene mutations of a representative p2gR-TRI-A line. Dots, deleted bases. Highlighting denotes the degree of homology of the aligned fragments. The type of indel and the number of indels of the same type are indicated.
Mentions: Two vectors, p2gR-TRI-A and p2gR-TRI-B (Figure 5A), each carrying two gRNAs targeting three genes related to trichome development, were used to transform Arabidopsis. Both vectors contain the same gRNA (T2-ETC2), which targets ETC2 and possibly CPC, a much less favorable target (Figure 5A). The vectors also contain different gRNAs (T1A-TC or T1B-TC). The 18-bp target sequence in T1A-TC is reversely complementary to that in T1B-TC. Both T1A-TC and T1B-TC target the same two genes: TRY and CPC (Figure 5A). There is only one mismatch between the 20-nt target of T1A-TC gRNA and TRY or CPC and between that of T1B-TC and TRY, whereas there are two mismatches between that of T1B-TC and CPC (Figure 5A). For p2gR-TRI-A, more than 70% of the T1 transgenic plants displayed highly clustered trichomes (Figure 5B,C), as expected for try cpc double or try cpc etc2 triple mutant plants [47]. For p2gR-TRI-B, less than 10% of the plants displayed the expected phenotypes, which suggests that T1B-TC has a much less favorable performance level than T1A-TC. Sequencing of the mutated alleles from a p2gR-TRI-B T1 transgenic line revealed that although the mutation efficiency of the TRY allele was more than 90%, that of CPC resulting from the same T1B-TC gRNA was only 42% (Additional file 1: Figure S1). By contrast, both CPC and TRY targeted by the same T1A-TC gRNA had similar mutation frequencies (greater than 90%), regardless of the fact that there were different PAMs between the two target sites (Figure 5A, D). These results suggest that the two mismatches might explain the poor performance of T1B-TC, although the two mismatches are located at the 5’-end of the gRNA. Furthermore, when there were three mismatches between the 20-nt target sequence of T2-ETC2 gRNA and the targeted gene CPC (Figure 5A), no mutation was detectable in more than 100 clones from the p2gR-TRI-A transgenic line. By contrast, ETC2 from the same T2-ETC2 gRNA had a mutation efficiency of 72% (Figure 5D). These results indicate that in planta, the CRISPR/Cas9 system has high sequence specificity, and two or more mismatches can greatly reduce the targeting efficiency and off-target effects, especially when a mismatch is near the 3′-end of the 20-nt target of a gRNA.Figure 5

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