Journal: Communications Biology

Article Title: A non-canonical sensing pathway mediates Plasmodium adaptation to amino acid deficiency

doi: 10.1038/s42003-023-04566-y

Figure Lengend Snippet: a , b Time course analysis of eIF2α phosphorylation status upon AA starvation of sorbitol-synchronized Pf 3D7 WT, eIK2, eIK1 and nek4 kinase knockout mutants (ring-stage window of ±8 h). Parasites were cultured in ( a ) MetS and MetD media or ( b ) IleS and IleD media. Starvation pulses were performed for 6 h at the ring- (R) or schizont- (Schiz) stage. Parasite proteins were extracted and probed for the phosphorylation of the translation initiation factor 2α (P-eIF2α) at the serine residue 51. Representative phospho-western blots and bar graphs quantifying phosphorylation levels of eIF2α, in ring- and schizont-stage parasites, relative to levels in ring-stage parasites growing under control conditions (MetS or IleS, respectively). Bars represent the mean of eIF2α-P levels relative to hsp70 levels ± SEM. c Schematic model for parasite’s adaptive response to AA depletion during intraerythrocytic development. Plasmodium parasites sense and actively respond to fluctuations in the availability of two distinct AAs: Ile and Met. Such response is heterogeneous as it depends on the AA that is depleted yet, it implies a common pathway comprising the phosphorylation of eIF2α by two distinct kinases, eIK1 and eIK2, which are temporally regulated at distinct stages of the life cycle, allowing parasites to fine-tune replication and development accordingly. This response leads to decreased replication, entry into a hibernation-like state and ultimately, extended survival.

Article Snippet: Primary antibodies used for fluorescence microscopy include: rabbit α HA (C29F4, Cell Signaling Technology) and mouse α Pb hsp70 (produced in house).

Techniques: Phospho-proteomics, Knock-Out, Cell Culture, Residue, Western Blot, Control

Journal: Cell reports

Article Title: Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

doi: 10.1016/j.celrep.2022.111226

Figure Lengend Snippet: (A) Rationale for Cas13d autoregulation. Overexpression likely produces many activated Cas13d:gRNA complexes for each CUG n target RNA. Minimizing Cas13d expression may greatly reduce collateral activity with minimal loss of on-target activity. (B) Description of GENO for regulation of Cas13d expression. In GENO, the pre-crRNA is placed in a UTR of the Cas13d mRNA, causing cleavage and degradation during crRNA processing. (C) Simulation of steady-state concentration of Cas13d:gRNA complex in GENO (solid line) and unregulated (dashed lines) conditions as a function of Cas13d transcription rate ( r t , horizontal axis) and gRNA transcription rate in the unregulated design ( r t ,gRNA , isolines). Autoregulation efficiency ƞ GENO is annotated (see ). Translation rate constant k T = 8.3 3 10 −3 protein mRNA −1 s −1 , crRNA processing rate constant k proc = 1.7 3 10 −7 cell mol −1 s −1 . (D) Simulated Cas13d:gRNA concentration as a function of k T (horizontal axis) and k proc (isolines). r t = 0.02 mRNA s −1 , r t ,gRNA = 0.96 RNA s −1 . (E) Fluorescent western blot comparing Cas13d protein expression from unregulated and GENO plasmids transfected in HeLa. Cas13d is visualized with α-HA antibody (green), and HSP70 is included as a control (pink). (F) Quantification of western blot. Error bars show SD. n = 3. ***p < 0.001, two-tailed t test. (G) Ratio of CUG 480 RNA to GAPDH mRNA after targeting CUG 480 with unregulated or GENO-Cas13d in HeLa, normalized to NT. n = 3 transfections, averaged across two qPCR replicates. Error bars show SEM of Δ C q propagated to the plotted ratio. *p < 0.05, one-tailed t test of Δ C q . (H) mCherry fluorescence relative to NT 20 h after transfection with unregulated (purple) and GENO (green) Cas13d and CUG-1 gRNA plasmids, CUG 480 target, and mCherry, as a function of mass of Cas13d plasmid transfected. Error bars show SD. n = 3. Logistic fit functions shown as solid lines. **p < 0.01, two-tailed t test. (I) MBNL1 exon 5 ψ measured by RT-PCR. Mass of Cas13d plasmid transfected matches the condition highlighted in (H). Error bars show SD. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t test.

Article Snippet: Mouse monoclonal α-HSP70 (5A5) , Invitrogen , Cat#MA3007; RRID:AB_325455.

Techniques: Over Expression, Expressing, Activity Assay, Concentration Assay, Western Blot, Transfection, Two Tailed Test, One-tailed Test, Fluorescence, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: Cell reports

Article Title: Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

doi: 10.1016/j.celrep.2022.111226

Figure Lengend Snippet: (A) Fluorescent western blot of Cas13d in AAV-transduced DM1 patient-derived myoblasts. Cas13d is visualized with α-HA antibody (green), and HSP70 is included as a control (pink). Protein ladder (L) is shown. (B) Representative 20× confocal images of AAV-treated DM1 myoblasts stained for Cas13d mRNA (HCR FISH, magenta) and protein (α-HA IF, yellow). Nuclei stained with DAPI (cyan). Scale bars, 10 μm. (C) Mean nuclear intensity of Cas13d HCR FISH in unregulated and GENO NT conditions. Dots represent nuclei, black line indicates median. n > 33 nuclei, >3 images. ***p < 0.001, two-sided Mann-Whitney U test. Gray line indicates mean baseline nuclear FISH signal in PBS-treated myoblasts, and gray shaded region indicates SD. n = 26 nuclei, 3 images. (D) Mean nuclear intensity of α-HA IF in unregulated and GENO NT conditions. Dots represent nuclei, black line indicates median. n > 33 nuclei, >3 images. ***p < 0.001, two-sided Mann-Whitney U test. Gray line and shaded region calculated as in (C). (E) Representative 40× confocal images of AAV-treated DM1 myoblasts stained for Cas13d mRNA (HCR FISH, magenta) and CUG n RNA (CAG 10 FISH probe, grayscale). Nuclei stained with DAPI (cyan). Scale bars, 10 μm. (F) Mean nuclear intensity of CUG n FISH in GENO targeting and NT conditions. Dots represent nuclei, black line indicates median. n > 43 nuclei, 21 images. *p < 0.05, one-sided Mann-Whitney U test. (G) Cumulative distribution function (c.d.f.) of FISH spot intensity of individual CUG n RNA foci in GENO targeting (magenta, solid) and NT (gray, solid) conditions. PBS-treated condition is also shown (gray, dashed). n > 745 spots. ***p < 0.001, two-sided Mann-Whitney U test.

Article Snippet: Mouse monoclonal α-HSP70 (5A5) , Invitrogen , Cat#MA3007; RRID:AB_325455.

Techniques: Western Blot, Derivative Assay, Staining, MANN-WHITNEY

Journal: Cell reports

Article Title: Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

doi: 10.1016/j.celrep.2022.111226

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal α-HSP70 (5A5) , Invitrogen , Cat#MA3007; RRID:AB_325455.

Techniques: Recombinant, Staining, Transfection, Electron Microscopy, Protease Inhibitor, Blocking Assay, Viability Assay, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Magnetic Beads, shRNA, Clone Assay, Software, Generated, Fluorescence, Microscopy

Journal: Cell reports

Article Title: Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

doi: 10.1016/j.celrep.2022.111226

Figure Lengend Snippet: (A) Rationale for Cas13d autoregulation. Overexpression likely produces many activated Cas13d:gRNA complexes for each CUG n target RNA. Minimizing Cas13d expression may greatly reduce collateral activity with minimal loss of on-target activity. (B) Description of GENO for regulation of Cas13d expression. In GENO, the pre-crRNA is placed in a UTR of the Cas13d mRNA, causing cleavage and degradation during crRNA processing. (C) Simulation of steady-state concentration of Cas13d:gRNA complex in GENO (solid line) and unregulated (dashed lines) conditions as a function of Cas13d transcription rate ( r t , horizontal axis) and gRNA transcription rate in the unregulated design ( r t ,gRNA , isolines). Autoregulation efficiency ƞ GENO is annotated (see ). Translation rate constant k T = 8.3 3 10 −3 protein mRNA −1 s −1 , crRNA processing rate constant k proc = 1.7 3 10 −7 cell mol −1 s −1 . (D) Simulated Cas13d:gRNA concentration as a function of k T (horizontal axis) and k proc (isolines). r t = 0.02 mRNA s −1 , r t ,gRNA = 0.96 RNA s −1 . (E) Fluorescent western blot comparing Cas13d protein expression from unregulated and GENO plasmids transfected in HeLa. Cas13d is visualized with α-HA antibody (green), and HSP70 is included as a control (pink). (F) Quantification of western blot. Error bars show SD. n = 3. ***p < 0.001, two-tailed t test. (G) Ratio of CUG 480 RNA to GAPDH mRNA after targeting CUG 480 with unregulated or GENO-Cas13d in HeLa, normalized to NT. n = 3 transfections, averaged across two qPCR replicates. Error bars show SEM of Δ C q propagated to the plotted ratio. *p < 0.05, one-tailed t test of Δ C q . (H) mCherry fluorescence relative to NT 20 h after transfection with unregulated (purple) and GENO (green) Cas13d and CUG-1 gRNA plasmids, CUG 480 target, and mCherry, as a function of mass of Cas13d plasmid transfected. Error bars show SD. n = 3. Logistic fit functions shown as solid lines. **p < 0.01, two-tailed t test. (I) MBNL1 exon 5 ψ measured by RT-PCR. Mass of Cas13d plasmid transfected matches the condition highlighted in (H). Error bars show SD. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t test.

Article Snippet: Blocking buffer was removed and replaced with primary antibody solution (rabbit α-HA (C29F4, 1:1000, Cell Signaling Technology) and mouse α-HSP70 (5A5, 1:500, Invitrogen) in 5 mL SEA BLOCK +0.2% Tween 20 (Fisher Bioreagents, #BP152–1)), and the membrane was placed in a 4°C room on a rocking mixer overnight.

Techniques: Over Expression, Expressing, Activity Assay, Concentration Assay, Western Blot, Transfection, Two Tailed Test, One-tailed Test, Fluorescence, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: Cell reports

Article Title: Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

doi: 10.1016/j.celrep.2022.111226

Figure Lengend Snippet: (A) Fluorescent western blot of Cas13d in AAV-transduced DM1 patient-derived myoblasts. Cas13d is visualized with α-HA antibody (green), and HSP70 is included as a control (pink). Protein ladder (L) is shown. (B) Representative 20× confocal images of AAV-treated DM1 myoblasts stained for Cas13d mRNA (HCR FISH, magenta) and protein (α-HA IF, yellow). Nuclei stained with DAPI (cyan). Scale bars, 10 μm. (C) Mean nuclear intensity of Cas13d HCR FISH in unregulated and GENO NT conditions. Dots represent nuclei, black line indicates median. n > 33 nuclei, >3 images. ***p < 0.001, two-sided Mann-Whitney U test. Gray line indicates mean baseline nuclear FISH signal in PBS-treated myoblasts, and gray shaded region indicates SD. n = 26 nuclei, 3 images. (D) Mean nuclear intensity of α-HA IF in unregulated and GENO NT conditions. Dots represent nuclei, black line indicates median. n > 33 nuclei, >3 images. ***p < 0.001, two-sided Mann-Whitney U test. Gray line and shaded region calculated as in (C). (E) Representative 40× confocal images of AAV-treated DM1 myoblasts stained for Cas13d mRNA (HCR FISH, magenta) and CUG n RNA (CAG 10 FISH probe, grayscale). Nuclei stained with DAPI (cyan). Scale bars, 10 μm. (F) Mean nuclear intensity of CUG n FISH in GENO targeting and NT conditions. Dots represent nuclei, black line indicates median. n > 43 nuclei, 21 images. *p < 0.05, one-sided Mann-Whitney U test. (G) Cumulative distribution function (c.d.f.) of FISH spot intensity of individual CUG n RNA foci in GENO targeting (magenta, solid) and NT (gray, solid) conditions. PBS-treated condition is also shown (gray, dashed). n > 745 spots. ***p < 0.001, two-sided Mann-Whitney U test.

Article Snippet: Blocking buffer was removed and replaced with primary antibody solution (rabbit α-HA (C29F4, 1:1000, Cell Signaling Technology) and mouse α-HSP70 (5A5, 1:500, Invitrogen) in 5 mL SEA BLOCK +0.2% Tween 20 (Fisher Bioreagents, #BP152–1)), and the membrane was placed in a 4°C room on a rocking mixer overnight.

Techniques: Western Blot, Derivative Assay, Staining, MANN-WHITNEY

Journal: Cell reports

Article Title: Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

doi: 10.1016/j.celrep.2022.111226

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Blocking buffer was removed and replaced with primary antibody solution (rabbit α-HA (C29F4, 1:1000, Cell Signaling Technology) and mouse α-HSP70 (5A5, 1:500, Invitrogen) in 5 mL SEA BLOCK +0.2% Tween 20 (Fisher Bioreagents, #BP152–1)), and the membrane was placed in a 4°C room on a rocking mixer overnight.

Techniques: Recombinant, Staining, Transfection, Electron Microscopy, Protease Inhibitor, Blocking Assay, Viability Assay, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Magnetic Beads, shRNA, Clone Assay, Software, Generated, Fluorescence, Microscopy