7b sgrna plasmids for nhej  (Addgene inc)

Journal: bioRxiv

Article Title: The N-terminal region of malaria vaccine candidate Plasmodium falciparum asparagine-rich merozoite antigen is immunodominant and targeted by polyreactive antibodies

doi: 10.64898/2025.12.11.693633

Figure Lengend Snippet: A) The percentage of IgM + and IgG + B cells with confirmed antigen-specificity obtained for PfARMA and four other merozoite proteins. B) Schematic of the predicted structural features of PfARMA. The asparagine-rich repeat region is shown in green. The six 100-amino acid (aa) long peptides with 50 aa overlap covering intrinsically disordered region (IDR) 1 are indicated below. C) The number of anti-PfARMA hmAbs targeting each of the three indicated regions of the protein. D) Percentage growth inhibition of P. falciparum strain 3D7 in in vitro cultures in the presence of hmAbs 5.134 and 4.104. Data points represent the average of three biological replicates, with the error bars showing the standard deviation. E) Dot-blot analysis showing reactivity of hmAb 5.134 to six IDR1 peptides and a negative control (NC) protein (PfAMA1). As a positive control for the presence of peptide and control protein, a second dot blot was stained in parallel with an anti-His antibody (bottom). F) The percentage of asparagine residues in overlapping 62-aa peptides covering the full P. falciparum ( Pf ) strain 3D7 proteome in the PhIP-seq library (n = 129,411) and among peptides bound by hmAb 5.134 (n = 303). Center line, median; box limits, upper and lower quartiles; whiskers, min/max values; +, mean. The difference between groups was tested using an unpaired student’s t-test. **** P < 0.0001.

Article Snippet: The plasmid encoding full-length PfARMA (PF3D7_1136200-bio, Addgene #47730) was modified to introduce a 6× His tag at the C-terminus.

Techniques: Inhibition, In Vitro, Standard Deviation, Dot Blot, Negative Control, Positive Control, Control, Staining

Journal: bioRxiv

Article Title: The N-terminal region of malaria vaccine candidate Plasmodium falciparum asparagine-rich merozoite antigen is immunodominant and targeted by polyreactive antibodies

doi: 10.64898/2025.12.11.693633

Figure Lengend Snippet: A) Immunofluorescence images of segmented schizonts stained with Hoechst 33342 (DNA), hmAb 5.134 against PfARMA and an antibody against a second merozoite protein (protein X), indicated on the left. Scale bar denotes 5 µm. B) Colocalization analysis of PfARMA with merozoite protein X using five different parasites for each pairwise analysis. The positive control (pos ctrl) was performed using anti-PfRAP1 primary antibody and a mix of two secondary antibodies conjugated to different fluorophores. Top: Pearson’s correlation, middle: Manders’ coefficient 1 (fraction of second protein signal that overlaps with PfARMA signal), bottom: Manders’ coefficient 2 (fraction of PfARMA signal that overlaps with the second protein signal). Differences in correlation coefficients tested for statistical significance using a Kruskal-Wallis test, followed by comparisons between all pairs of groups using Dunn’s post-hoc test, which reports P values that have been corrected for multiple comparisons (n = 21). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The plasmid encoding full-length PfARMA (PF3D7_1136200-bio, Addgene #47730) was modified to introduce a 6× His tag at the C-terminus.

Techniques: Immunofluorescence, Staining, Positive Control

Journal: bioRxiv

Article Title: The N-terminal region of malaria vaccine candidate Plasmodium falciparum asparagine-rich merozoite antigen is immunodominant and targeted by polyreactive antibodies

doi: 10.64898/2025.12.11.693633

Figure Lengend Snippet: Plasma samples from P. falciparum -unexposed U.S. donors (U, n = 15) and P. falciparum -exposed individuals with low (L, n = 46), moderate (M, n = 40), and high (H, n = 54) immunity to malaria were measured in duplicate, with the average of both readings shown for ( A ) PfMSP1 and full-length PfARMA and ( B ) fragments of PfARMA. PfMSP1 was included as a control for P. falciparum exposure and is therefore shown on a gray background. The groups with low and medium immunity were matched for age and P. falciparum exposure history. The dashed horizontal line indicates the cutoff for seropositivity (three standard deviations above the average for the entire P. falciparum -naïve group, or for the subset of P. falciparum -naïve individuals without IgG reactivity for full-length PfARMA and IDR1). Center line, median; box limits, upper and lower quartiles; whiskers, min/max values; +, mean. Differences between groups were tested using a Kruskal-Wallis test, followed by comparisons between all pairs of groups using Dunn’s post-hoc test, which reports P values that have been corrected for multiple comparisons (n = 6). Within each graph, groups sharing the same letter are not statistically significantly different from each other, while groups with different letters are statistically significantly different (P < 0.05). No statistically significant differences were observed between the groups with low and medium immunity. IDR, intrinsically disordered region; FD, folded domain.

Article Snippet: The plasmid encoding full-length PfARMA (PF3D7_1136200-bio, Addgene #47730) was modified to introduce a 6× His tag at the C-terminus.

Techniques: Clinical Proteomics, Control

Journal: bioRxiv

Article Title: The N-terminal region of malaria vaccine candidate Plasmodium falciparum asparagine-rich merozoite antigen is immunodominant and targeted by polyreactive antibodies

doi: 10.64898/2025.12.11.693633

Figure Lengend Snippet: A) Plasma samples were obtained from P. falciparum -unexposed individuals living in the U.S. who were convalescent COVID-19 patients (n = 7), healthy blood donors (n = 8), systemic lupus erythematosus (SLE) patients (n = 10), or rheumatoid arthritis (RA) patients (n = 8). In all plots, the horizontal dashed line indicates the cut-off for reactivity to PfARMA (MFI = 2.6 × 10 3 ), which equals the average MFI + three standard deviations of reactivity to the other six merozoite antigens among samples from non-autoimmune donors (convalescent COVID-19 patients and healthy blood donors). Differences in reactivity to the various merozoite antigens were tested for statistical significance using a Kruskal-Wallis test, followed by comparisons between PfARMA and all other antigens using Dunn’s post-hoc test, which reports P values that have been corrected for multiple comparisons (n = 6). ** P < 0.01; *** P < 0.001. B) Dose-dependent reactivity of IgG purified from plasma of a P. falciparum -naïve U.S. donor with high anti-PfARMA IgG reactivity to PfMSP1, full-length PfARMA, and fragments of PfARMA, as measured by ELISA. IDR, intrinsically disordered region; FD, folded domain. C) Total plasma IgG concentration plotted against PfARMA IgG reactivity for P. falciparum -naïve individuals and P. falciparum -exposed individuals. The line shows the best-fit using simple linear regression, with 95% confidence intervals. D) Reactivity of recombinant IgG1, IgG2, IgG3, and IgG4 with specificity for a different P. falciparum antigen to PfARMA and PfMSP1.

Article Snippet: The plasmid encoding full-length PfARMA (PF3D7_1136200-bio, Addgene #47730) was modified to introduce a 6× His tag at the C-terminus.

Techniques: Clinical Proteomics, Purification, Enzyme-linked Immunosorbent Assay, Concentration Assay, Recombinant

Journal: bioRxiv

Article Title: The N-terminal region of malaria vaccine candidate Plasmodium falciparum asparagine-rich merozoite antigen is immunodominant and targeted by polyreactive antibodies

doi: 10.64898/2025.12.11.693633

Figure Lengend Snippet: A) Overview of the affinity purification of autoantibodies and anti-PfARMA IgG from plasma IgG from P. falciparum -naïve and P. falciparum -exposed individuals. B) Reactivity of affinity-purified autoantibodies (AAb) and the flowthrough (FT) fraction from plasma IgG of P. falciparum -naïve individuals, both tested at 20 µg/mL, to a panel of seven merozoite antigens (left) and PfARMA fragments (right). The average fold change in MFI in the autoantibody fractions relative to the flowthrough fractions is indicated in the top. C) Heatmap showing binding of anti-PfARMA plasma IgG isolated from P. falciparum -naïve and P. falciparum -exposed individuals by affinity purification, as well as anti-PfARMA hmAbs, to (macro)molecules with properties often recognized by auto- or polyreactive antibodies, measured by ELISA. Values are color-coded based on the optical density (OD) after subtraction of the background signal. OD = 0.4 was used as the cutoff for binding. DNP-BSA, dinitrophenol-bovine serum albumin; CFP, cyan-fluorescent protein; KLH, keyhole limpet hemocyanin; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.

Article Snippet: The plasmid encoding full-length PfARMA (PF3D7_1136200-bio, Addgene #47730) was modified to introduce a 6× His tag at the C-terminus.

Techniques: Affinity Purification, Clinical Proteomics, Binding Assay, Isolation, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The N-terminal region of malaria vaccine candidate Plasmodium falciparum asparagine-rich merozoite antigen is immunodominant and targeted by polyreactive antibodies

doi: 10.64898/2025.12.11.693633

Figure Lengend Snippet: A) PfARMA peptides recognized by plasma IgG from P. falciparum -exposed and P. falciparum -naïve individuals. All peptides were located in intrinsically disordered region (IDR) 1 and overlapped either a negatively charged patch (shown in red) or the asparagine-rich repeat region (shown in green). B) Non-PfARMA-derived peptides that were bound by hmAb 5.134 and one or more plasma IgG samples. The red shading indicates the percentage of asparagine residues in the peptides. Absence of reactivity to peptides is indicated with gray shading.

Article Snippet: The plasmid encoding full-length PfARMA (PF3D7_1136200-bio, Addgene #47730) was modified to introduce a 6× His tag at the C-terminus.

Techniques: Clinical Proteomics, Derivative Assay