Dataset Information

A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in Arabidopsis.

ABSTRACT: The CRISPR/Cas9 genome-editing system has emerged as a popular powerful tool for biological research. However, the process of selecting efficiently edited Cas9-free plants is usually laborious and time consuming. Here, we demonstrated P2A to be the most efficient self-cleaving peptide for fusing Cas9 and GFP in Arabidopsis and then used Cas9-P2A-GFP to develop a novel CRISPR/Cas9 system. Additionally, a pair of isocaudomer restriction enzymes were selected to conveniently assemble multiple sgRNAs. In this system, the GFP fluorescence intensity in T1 transgenic plants indicates the expression level of the Cas9 protein, which correlates well with the editing efficiency. Furthermore, Cas9-free plants can be easily selected by examining GFP fluorescence in T2 transgenic plants. The efficient knockout of BRI1, BZR1 and BES1 demonstrated the robustness of our new system. Thus, we designed a novel CRISPR/Cas9 system that can generate Cas9-free multiplex mutants efficiently in Arabidopsis and possibly in other plant species.

SUBMITTER: Wang J

PROVIDER: S-EPMC9584096 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in <i>Arabidopsis</i>.

Wang Jiajun J Chen Haodong H

aBIOTECH 20191120 1

The CRISPR/Cas9 genome-editing system has emerged as a popular powerful tool for biological research. However, the process of selecting efficiently edited Cas9-free plants is usually laborious and time consuming. Here, we demonstrated P2A to be the most efficient self-cleaving peptide for fusing Cas9 and GFP in <i>Arabidopsis</i> and then used Cas9-P2A-GFP to develop a novel CRISPR/Cas9 system. Additionally, a pair of isocaudomer restriction enzymes were selected to conveniently assemble multipl ...[more]

PMID: 36305009

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Project description:BackgroundMultiplex CRISPR-Cas9-based genome editing is an efficient method for targeted disruption of gene function in plants. Use of CRISPR-Cas9 has increased rapidly in recent years and is becoming a routine method for generating single and higher order Arabidopsis thaliana mutants. Low entry, reliable assembly of CRISPR/Cas9 vectors and efficient mutagenesis is necessary to enable a maximum of researchers to break through the genetic redundancy within plant multi-gene families and allow for a plethora of gene function studies that have been previously unachievable. It will also allow routine de novo generation of mutations in ever more complex genetic backgrounds that make introgression of pre-existing alleles highly cumbersome.ResultsTo facilitate rapid and efficient use of CRISPR/Cas9 for Arabidopsis research, we developed a CRISPR/Cas9-based toolbox for generating mutations at multiple genomic loci, using two-color fluorescent seed selection. In our system, up-to eight gRNAs can be routinely introduced into a binary vector carrying either a FastRed, FastGreen or FastCyan fluorescent seed selection cassette. FastRed and FastGreen binary vectors can be co-transformed as a cocktail via floral dip to introduce sixteen gRNAs at the same time. The seeds can be screened either for red or green fluorescence, or for the presence of both colors. Importantly, in the second generation after transformation, Cas9 free plants are identified simply by screening the non-fluorescent seeds. Our collection of binary vectors allows to choose between two widely-used promoters to drive Cas enzymes, either the egg cell-specific (pEC1.2) from A. thaliana or the constitutive promoter from Petroselinum crispum (PcUBi4-2). Available enzymes are "classical" Cas9 codon-optimized for A. thaliana and a recently reported, intron-containing version of Cas9 codon-optimized for Zea mays, zCas9i. We observed the highest efficiency in producing knockout phenotypes by using intron-containing zCas9i driven under egg-cell specific pEC1.2 promoter. Finally, we introduced convenient restriction sites flanking promoter, Cas9 and fluorescent selection cassette in some of the T-DNA vectors, thus allowing straightforward swapping of all three elements for further adaptation and improvement of the system.ConclusionA rapid, simple and flexible CISPR/Cas9 cloning system was established that allows assembly of multi-guide RNA constructs in a robust and reproducible fashion, by avoiding generation of very big constructs. The system enables a flexible, fast and efficient screening of single or higher order A. thaliana mutants.

Dataset Information

A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in Arabidopsis.

Publications

A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in <i>Arabidopsis</i>.

Similar Datasets

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