Single-step assembly of a dual sgRNA expression vector and efficient genomic deletion.

(A) Schematic of the dual sgRNA expression vector designed for single-step assembly. The yellow highlights indicate the BpiI sites, and the green and orange letters show the target sequences of the sgRNAs and the overhangs for the Golden Gate cloning, respectively. The BpiI sites in both the first and second sgRNA sites generate different overhangs after digestion. act15, act15 promoter; act8, act8 terminator; tRNA, isoleucine tRNA; act6, act6 promoter. (B) Correct insertion of the B1-2 and T1 sgRNA sequences into pTM1544 via one-step cloning. Correct insertion was confirmed via colony PCR. The B1-2 sequence was inserted as the first target (F), and T1 was inserted as the second target (S). (C) Genomic deletions generated via transient introduction of pTM1544. PCR products produced using primers flanking the target sites are shown. The arrowhead indicates product size found in the control (the AX2 genome).</p

Double nicking-mediated precise genome editing via HDR.

(A) Schematic illustration of the precise genome editing approach using paired Cas9 nickases and single-stranded oligonucleotides (ssODNs). Point mutations and deletions are marked by orange broken lines and asterisk, respectively. (B) The sequences used as the ssODN templates. A 66 nt deletion and EcoRI restriction site are shown in orange. Point mutations are indicated by orange lowercase letters and the BamHI site is underlined. (C, D) PCR and restriction enzyme analysis of individual clones. PCR products before (-) and after digestion with the appropriate restriction enzyme (+) are shown. (E, F) The wild-type and mutated pkaC sequences. Target sequences are blue and mutations are in red.</p

List of oligonucleotides used to generate the sgRNA vectors.

List of oligonucleotides used to generate the sgRNA vectors.</p

Long genomic deletions generated via use of paired Cas9 nickases.

(A) Target sites in the pkaC gene locus. The position of each target site is indicated by an arrowhead. The arrows indicate the locations of the PCR primers used to detect the deletions. The box in green represents the protein kinase domain predicted by UniProt. (B) Analysis of the deletion efficiencies of paired Cas9 nickases. A total of 22 independent clones were isolated from individual transformations and scored for the presence of deletions via PCR. The error bars show the standard error of the mean based on three independent transformations. (C) Representative genomic deletions detected by PCR. The arrowhead indicates product size found in the control (the AX2 genome). Clone numbers are shown in red and black, and indicate obvious and unapparent deletions, respectively. (D) Aggregation phenotypes of the mutants. (E) Representative DNA sequences of the target region. The PAM sequences are shown by green underscores. The sgRNA-matching sequences are shown in blue. The mutated nucleotides are in red, and the numbers in parentheses indicate the number of deleted nucleotides.</p

CRISPR/Cas9-mediated generation of deletion mutations in <i>Dictyostelium</i>.

(A) Schematic overview of the CRISPR vectors designed to generate deletion mutations. sgRNA sequences were synthesised as pairs of oligonucleotides and integrated into the vectors using the Golden Gate assembly method. act15, act15 promoter; act8, act8 terminator; tRNA, isoleucine tRNA. (B) CRISPR-mediated deletion efficiencies. Single clones grown on SM agar plates were randomly selected and tested for deletions via PCR amplification of the target region. The error bars show the standard error of the mean based on three independent biological repeats. (C) Genomic deletions in pTM1331-expressing cells. PCR products generated using primers flanking the target sites are shown. A control reaction using AX2 cells is shown in the rightmost lane. Red arrowhead indicates PCR products found in the control. (D) Aggregation phenotypes of the mutants shown in Fig 1C. Single clones were seeded on a bacterial lawn and imaged after 4 days with a stereoscopic microscope. (E) The sequencing results of the deletion regions. The wild-type (WT) sequence of pkaC and six independent mutated sequences are shown. The sequence used as the target is shown in blue and the PAM sequences are shown by green underscores. The mutated nucleotides are in red, and the numbers in parentheses indicate the number of deleted nucleotides.</p

Data_Sheet_1_CRISPR Toolbox for Genome Editing in Dictyostelium.PDF

The development of new techniques to create gene knockouts and knock-ins is essential for successful investigation of gene functions and elucidation of the causes of diseases and their associated fundamental cellular processes. In the biomedical model organism Dictyostelium discoideum, the methodology for gene targeting with homologous recombination to generate mutants is well-established. Recently, we have applied CRISPR/Cas9-mediated approaches in Dictyostelium, allowing the rapid generation of mutants by transiently expressing sgRNA and Cas9 using an all-in-one vector. CRISPR/Cas9 techniques not only provide an alternative to homologous recombination-based gene knockouts but also enable the creation of mutants that were technically unfeasible previously. Herein, we provide a detailed protocol for the CRISPR/Cas9-based method in Dictyostelium. We also describe new tools, including double knockouts using a single CRISPR vector, drug-inducible knockouts, and gene knockdown using CRISPR interference (CRISPRi). We demonstrate the use of these tools for some candidate genes. Our data indicate that more suitable mutants can be rapidly generated using CRISPR/Cas9-based techniques to study gene function in Dictyostelium.</p