ppbu6 hdhfr yfcu cas9  (Addgene inc)

Journal: bioRxiv

Article Title: A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei

doi: 10.1101/2024.04.20.590404

Figure Lengend Snippet: ( A ) Schematic of pPbU6-hdhfr/yfcu-Cas9 with the homology regions (HR) of the repair template delivered on the same plasmid (one-plasmid approach) or on a PCR product (PCR-template approach) to generate the rap2/3 -3xHA parasite line. The effect on transfection efficiency was compared by both ( i ) substituting the P. yoelii U6 for the P. berghei U6 promoter (not significant) and ( ii ) different homology arm length (p = 0.0068). ( iii ) The relationship between homology arm length and transfection efficiency was examined by plotting homology arm length against the day parasitemia reached >0.5%. One or two-way ANOVAs were performed to compare transfection efficiencies from two biological replicates per condition. All parasitemia counts can be found in Table S2 . ( B ) The expression of RAP2/3-3xHA was confirmed by Western blot following transfection with the pPbU6-hdhfr/yfcu-Cas9 vector using ∼500 bp HR. Wild type parasites were used as a negative control for the HA tag and the anti-EXP1 was used as a loading control. Full-length Western blots are shown in Fig. S2 . ( C ) Growth assay of the PbCas9 line that constitutively expresses FLAG-Cas9 under the hsp70 promoter with the PbGIMO mother line used as a control. Error bars = SD for three biological replicates. ( D ) FLAG-Cas9 expression was confirmed using Western blot, where FLAG detects Cas9 and anti-EXP1 was used as loading control. PbGIMO was used as negative control for the FLAG tag. ( E ) Immunofluorescence assay demonstrates that FLAG-Cas9 is expressed and localised to the nucleus. FLAG detects Cas9 and 4′,6-diamidino-2-phenylindole (DAPI) stains the nucleus. BF = Bright-field. The PbGIMO mother line was used as a negative control. Scale bar = 5 µm. ( F ) Transfection efficiency of the pPbU6-hdhfr/yfcu rap2/3 -3xHA vector into the PbCas9 line was assessed using the one-plasmid versus the PCR-template approach. Error bars = SD for two biological replicates.

Article Snippet: The pPbU6-hdhfr/yfcu plasmid and the synthetic fragment were digested with Bsm BI and Aat II (NEB) and ligated into the vector with T4 ligase (NEB).

Techniques: Plasmid Preparation, Transfection, Expressing, Western Blot, Negative Control, Growth Assay, FLAG-tag, Immunofluorescence

Journal: Nucleic Acids Research

Article Title: A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei

doi: 10.1093/nar/gkaf005

Figure Lengend Snippet: Characterization of pPbU6-hdhfr/yfcu vector systems and the PbCas9 mother line. ( A ) Schematic of pPbU6-hdhfr/yfcu-Cas9 with the HRs of the repair template delivered on the same plasmid (one-plasmid approach) or on a PCR product (PCR-template approach) to generate the rap2/3 –3×HA parasite line. The effect on transfection efficiency was compared by both ( i ) substituting the PyU6 for the PbU6 promoter (not significant) and ( ii ) different homology arm length ( p = 0.0068). ( iii ) The relationship between homology arm length and transfection efficiency was examined by plotting homology arm length against the day parasitemia reached >0.5%. One or two-way ANOVAs were performed to compare transfection efficiencies from two biological replicates per condition. The expression of RAP2/3–3×HA was confirmed by western blot following transfection with the pPbU6-hdhfr/yfcu-Cas9 vector using ∼500-bp HR. Wild-type parasites were used as a negative control for the HA tag and the anti-EXP1 was used as a loading control. ( C ) Growth assay of the PbCas9 line that constitutively expresses FLAG-Cas9 under the hsp70 promoter with the PbGIMO mother line used as a control. Error bars = SD for three biological replicates. ( D ) FLAG-Cas9 expression was confirmed using western blot, where FLAG detects Cas9 and anti-EXP1 was used as loading control. PbGIMO was used as a negative control for the FLAG tag. ( E ) IFA demonstrates that FLAG-Cas9 is expressed and localized to the nucleus. FLAG detects Cas9 and DAPI stains the nucleus. BF = Bright-field. The PbGIMO mother line was used as a negative control. Scale bar = 5 μm. ( F ) Transfection efficiency of the pPbU6-hdhfr/yfcu rap2/3 –3×HA vector into the PbCas9 line was assessed using the one-plasmid versus the PCR-template approach. Error bars = SD for two biological replicates. Full-length western blots are shown in and and parasitemia counts can be found in .

Article Snippet: When pPbU6-hdhfr/yfcu-Cas9 or pPbU6-hdhfr/yfcu was used, pyrimethamine (0.07 mg/ml, MP Biomedicals) was removed at day 5 post-transfection.

Techniques: Plasmid Preparation, Transfection, Expressing, Western Blot, Negative Control, Control, Growth Assay, FLAG-tag