Abstract
In the last 30 years, knockout of target genes via homologous recombination has been widely performed to clarify the physiological functions of proteins in Dictyostelium. As of late, CRISPR/Cas9-mediated genome editing has become a versatile tool in various organisms, including Dictyostelium, enabling rapid high-fidelity modification of endogenous genes. Here we reviewed recent progress in genome editing in Dictyostelium and summarised useful CRISPR vectors that express sgRNA and Cas9, including several microorganisms. Using these vectors, precise genome modifications can be achieved within 2–3 weeks, beginning with the design of the target sequence. Finally, we discussed future perspectives on the use of CRISPR/Cas9-mediated genome editing in Dictyostelium.
Highlights
In the last 30 years, knockout of target genes via homologous recombination has been widely performed to clarify the physiological functions of proteins in Dictyostelium
To analyse the functions of these homologous proteins, powerful molecular genetics approaches are available for Dictyostelium, including the expression of GFP- or epitope-tagged proteins, gene knockout via homologous recombination, insertional mutagenesis and gene silencing
Drawback of RNA polymerase III-dependent promoters lacks the ability to express in a tissue-specific manner to achieve tissue-specific genome editing, the ribozyme-based single-guide RNA (sgRNA) production can be used as tissue-specific promoters or promoters regulated by environmental signals because the primary transcripts are automatically processed to release sgRNAs
Summary
The social amoeba Dictyostelium is a microbial model organism widely used to understand cellular and developmental biology. Drawback of RNA polymerase III-dependent promoters lacks the ability to express in a tissue-specific manner to achieve tissue-specific genome editing, the ribozyme-based sgRNA production can be used as tissue-specific promoters or promoters regulated by environmental signals because the primary transcripts are automatically processed to release sgRNAs. TcPFR1, TcPFR2, TcGP72 human codon-optimised SpCas driven by ribosomal promoter. In the mutation detective PCR, the amplification efficiency at the Cas cleavage region is compared between CRISPR-mediated mutants and the control clone that is not targeted in the gene. Recognition of the appropriate target site by sgRNA in the CRISPR/Cas system is sufficiently specific, as it has been reported that cleavage activity is decreased when a single nucleotide mismatch is present within the sgRNA sequence in mammalian cells [54]. It cannot be said that there are no off-target effects in Dictyostelium, and the analysis of multiple mutants is recommended
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