Abstract
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.
Highlights
Multiplex CRISPR-Cas9-based genome editing is an efficient method for targeted disruption of gene function in plants
The first step includes generation of entry clones where single guide RNA (sgRNA) are introduced into a vector containing either the pU6 or pU3 promoter and guide RNA (gRNA) scaffold via a simple oligo annealing technique
This is a single tube reaction and only requires an annealed oligonucleotide pair to serve as the sgRNA molecule of choice to be introduced into a convenient BbsI restriction site (Fig. 1a; Additional file 1: Table S1)
Summary
Multiplex CRISPR-Cas9-based genome editing is an efficient method for targeted disruption of gene function in plants. Reliable assembly of CRISPR/Cas 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 allow routine de novo generation of mutations in ever more complex genetic backgrounds that make introgression of pre-existing alleles highly cumbersome. NHEJ occurs most often, is highly efficient but inaccurate, and tends to introduce errors in the form of small insertions or deletions that are usually sufficient to knock out the gene This technique is widely used to produce stable single and multiple mutants in various plant species. Grützner et al [20] reported that the addition of 13 introns into the Cas coding sequence in combination with two nuclear localization signals (NLS) led to higher accumulation of the Cas in the nucleus and significant improvement of editing efficiency
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