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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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Thetry cpc etc2triple mutant can be differentiated fromtry cpcdouble mutant. Representative triple and double mutants and the wild type are shown. The seeds were sown on MS plates, vernalized at 4°C for 3 days, and transferred to an illumination incubator and allowed to grow for 10 days. The seedlings were transplanted to soil and allowed to grow for 17 days before photographing. The triple and double mutants were segregated from A17 T1 lines.
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Fig6: Thetry cpc etc2triple mutant can be differentiated fromtry cpcdouble mutant. Representative triple and double mutants and the wild type are shown. The seeds were sown on MS plates, vernalized at 4°C for 3 days, and transferred to an illumination incubator and allowed to grow for 10 days. The seedlings were transplanted to soil and allowed to grow for 17 days before photographing. The triple and double mutants were segregated from A17 T1 lines.

Mentions: There were many different mutated alleles in a single transgenic plant (Figure 5D), which suggests that the CRISPR/Cas9 functioned after the division of fertilized eggs. To confirm that germline transmission of the mutations into T2 plants is possible, we examined the transmission of mutations of five p2gR-TRI-A T1 transgenic lines with strong phenotypes. Since Cas9 and gRNA are constitutively expressed in T2 transgenic lines, it is sometimes difficult to determine the sources of mutations, which might have arisen from germ cells or somatic cells. On the contrary, mutations from separated T2 nontransgenic plants must result from germline transmission of the mutations from T1 plants. Therefore, we focused on segregated nontransgenic T2 plants to simplify analysis of germline transmission of the mutations. The nontransgenic plants were identified by PCR counterselection with three primer pairs, including two for the hygromycin-resistance gene and one for Cas9. We determined the biallelic mutations for both TRY and CPC based on their clustered trichome phenotypes, finding that the TRY and CPC mutations were transmitted to T2 plants with high efficiency; 46.2%, 100%, 82.6%, 100%, and 100% of nontransgenic T2 plants derived from five T1 lines, respectively, were biallelic mutants for both TRY and CPC (Table 1). We first analyzed ETC2 mutations of nontransgenic T2 double mutant plants by directly sequencing PCR products or by sequencing DNA from different clones harboring the PCR products, and we then analyzed TRY and CPC mutations of etc2 mutants verified in the first step of analysis (Table 2). We found that the verified try cpc etc2 triple mutants could easily be differentiated from try cpc double mutants; the former plants were shorter than the latter and had upwardly curled leaves (Figure 6). Biallelic T2 mutants for ETC2 were segregated from only two T1 lines among the five lines examined (Table 1), demonstrating that the frequency of germline transmission of the ETC2 mutations into T2 plants was much lower than that of the TRY and CPC mutations. This result could be explained by the lower mutation frequencies of ETC2 in T1 plants.Table 1


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)

Thetry cpc etc2triple mutant can be differentiated fromtry cpcdouble mutant. Representative triple and double mutants and the wild type are shown. The seeds were sown on MS plates, vernalized at 4°C for 3 days, and transferred to an illumination incubator and allowed to grow for 10 days. The seedlings were transplanted to soil and allowed to grow for 17 days before photographing. The triple and double mutants were segregated from A17 T1 lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Thetry cpc etc2triple mutant can be differentiated fromtry cpcdouble mutant. Representative triple and double mutants and the wild type are shown. The seeds were sown on MS plates, vernalized at 4°C for 3 days, and transferred to an illumination incubator and allowed to grow for 10 days. The seedlings were transplanted to soil and allowed to grow for 17 days before photographing. The triple and double mutants were segregated from A17 T1 lines.
Mentions: There were many different mutated alleles in a single transgenic plant (Figure 5D), which suggests that the CRISPR/Cas9 functioned after the division of fertilized eggs. To confirm that germline transmission of the mutations into T2 plants is possible, we examined the transmission of mutations of five p2gR-TRI-A T1 transgenic lines with strong phenotypes. Since Cas9 and gRNA are constitutively expressed in T2 transgenic lines, it is sometimes difficult to determine the sources of mutations, which might have arisen from germ cells or somatic cells. On the contrary, mutations from separated T2 nontransgenic plants must result from germline transmission of the mutations from T1 plants. Therefore, we focused on segregated nontransgenic T2 plants to simplify analysis of germline transmission of the mutations. The nontransgenic plants were identified by PCR counterselection with three primer pairs, including two for the hygromycin-resistance gene and one for Cas9. We determined the biallelic mutations for both TRY and CPC based on their clustered trichome phenotypes, finding that the TRY and CPC mutations were transmitted to T2 plants with high efficiency; 46.2%, 100%, 82.6%, 100%, and 100% of nontransgenic T2 plants derived from five T1 lines, respectively, were biallelic mutants for both TRY and CPC (Table 1). We first analyzed ETC2 mutations of nontransgenic T2 double mutant plants by directly sequencing PCR products or by sequencing DNA from different clones harboring the PCR products, and we then analyzed TRY and CPC mutations of etc2 mutants verified in the first step of analysis (Table 2). We found that the verified try cpc etc2 triple mutants could easily be differentiated from try cpc double mutants; the former plants were shorter than the latter and had upwardly curled leaves (Figure 6). Biallelic T2 mutants for ETC2 were segregated from only two T1 lines among the five lines examined (Table 1), demonstrating that the frequency of germline transmission of the ETC2 mutations into T2 plants was much lower than that of the TRY and CPC mutations. This result could be explained by the lower mutation frequencies of ETC2 in T1 plants.Table 1

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