Abstract
CRISPR/Cas9 has emerged in various organisms as a powerful technology for targeted gene knockout; however, no reports of editing the Dictyostelium genome efficiently using this system are available. We describe here the application of CRISPR/Cas9-mediated gene modification in Dictyostelium. The endogenous tRNA-processing system for expressing sgRNA was approximately 10 times more effective than the commonly used U6 promoter. The resulting sgRNA affected the sub-nuclear localisation of Cas9, indicating that the expression level of sgRNA was sufficiently high to form Cas9 and sgRNA complexes within the nucleus. The all-in-one vector containing Cas9 and sgRNA was transiently expressed to generate mutants in five PI3K genes. Mutation detective PCR revealed the mutagenesis frequency of the individual genes to be between 72.9% and 100%. We confirmed that all five targeting loci in the four independent clones had insertion/deletion mutations in their target sites. Thus, we show that the CRISPR/Cas9 system can be used in Dictyostelium cells to enable efficient genome editing of multiple genes. Since this system utilises transient expression of the all-in-one vector, it has the advantage that the drug resistance cassette is not integrated into the genome and simple vector construction, involving annealing two oligo-DNAs.
Highlights
The simple amoeboid eukaryote Dictyostelium discoideum is a model system, that is used to study several biological processes such as growth, macropinocytosis, cell motility, chemotaxis, and signal transduction during development[1]
To compare the expression efficiency of the single-guide RNA (sgRNA) in Dictyostelium under the control of a different promoter, we constructed two sgRNA expression vectors derived from the U6 promoter or isoleucine tRNA
All gRNA transcripts were cleaved at the tRNA-sgRNA junction and a half of the clones were cleaved precisely without the addition of extra nucleotides (Fig. 1C)
Summary
The simple amoeboid eukaryote Dictyostelium discoideum is a model system, that is used to study several biological processes such as growth, macropinocytosis, cell motility, chemotaxis, and signal transduction during development[1]. Using the homologous recombination technique, a DNA template typically over 500 bp homology sequences, is designed to recombine at the genomic locus of interest Because many of these genes reveal overlapping functions, there is a high demand for the generation of multiple gene knockouts within a single cell. In the CRISPR/Cas[9] system, because a ~20 nucleotide target sequence is used to achieve site-specific DSB, www.nature.com/scientificreports/. These difficulties can be avoided in some cases. The simple procedure of CRISPR/Cas[9] has created a possibility of applying transcriptional activation/repression, epigenetic modification, genomic imaging and the targeting of multiple genes[16,17,18]. The insertion of sgRNA between tRNA sequences causes RNase P and RNase Z to recognise the cloverleaf structure and cleave at the 5′ and 3′ ends of tRNA, resulting in the production of mature sgRNAs21
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