engen sau cas9 nuclease  (New England Biolabs)

Journal: bioRxiv

Article Title: Astrocyte-derived MFG-E8 facilitates microglial synapse elimination in Alzheimer’s disease mouse models

doi: 10.1101/2024.08.31.606944

Figure Lengend Snippet: A) PCR product resolved on agarose gel after amplification of genomic DNA (gDNA) regions of the Mfge8 gene. These gDNA amplicons correspond to the cleavage regions of the sgRNAs. Primer 1-6 refers to the primer set used to amplify each region. Expected amplicon product lengths: primer 1, 275 bp; primer 2, 254 bp; primer 3, 323 bp; primer 4, 275 bp; primer 5, 380 bp; primer 6, 232 bp. B) PCR product resolved on agarose gel after amplification of 6 DNA templates for subsequent transcription of the sgRNA molecules. Each template corresponds to one sgRNA sequence. The arrow points at the expected amplicon length (121 bp). C) In vitro digestion assay resolved on agarose gel to assess the cleavage efficiency of each designed sgRNA. The reaction was performed with the sgRNA + Cas9 or as a control the Cas9 or sgRNA alone. Black arrows indicate the input gDNA length and the red arrows indicate the expected cleavage fragments. Only the three successful reactions are shown here. Expected fragment lengths: Set 1, 218 bp and 31 bp; Set 3, 206 bp and 90 bp, Set 6, 127 bp and 104 bp. D) Mfge8 gRNA- GfaABC 1 D -mCherry plasmid construct map. E) GfaABC 1 D -saCas9-HA plasmid construct map.

Article Snippet: A PCR reaction was performed using 100 nM of the purified RNA product, 200 ng of the amplified gDNA region product and 1 μM EnGen® Sau Cas9 (#M0654, NEB), according to the manufacturer, and running the reaction at 37°C for 1 hr, followed by 5 min at 75°C.

Techniques: Agarose Gel Electrophoresis, Amplification, Sequencing, In Vitro, Control, Plasmid Preparation, Construct

Journal: bioRxiv

Article Title: Astrocyte-derived MFG-E8 facilitates microglial synapse elimination in Alzheimer’s disease mouse models

doi: 10.1101/2024.08.31.606944

Figure Lengend Snippet: Viral CRISPR-saCas9 deletion of astrocytic MFG-E8 prevents microglia-Homer1 engulfment and excitatory loss in the NL-F KI hippocampus . A) Schematic of CRISPR-saCas9 paradigm to knock-down Mfge8 from hippocampal astrocytes with spatio-temporal control. Immunostaining for gRNA-mCherry (magenta) and Cas9-HA (green) constructs post-CRISPR-saCas9 injection. Scale bar = 5 nm. Representative image of MFG-E8 (white) immunoreactivity post-CRISPR-saCas9 injection. Scale bar = 1 nm. Inset highlights ROIs. mCherry injection zone drawn with white ROI on ImageJ. B) Representative images of gRNA-mCherry (red) and Cas9-HA (green) constructs with either GFAP (blue, left), NeuN (blue, middle) or Iba1 (blue, right) post-CRISPR-saCas9 injection. Scale bar = 30 μm. C) Quantification of the colocalized mCherry-labeled signal with GFAP + astrocytes, NeuN + neurons or Iba1 + microglia in the hippocampus using ImageJ shown as percentage of the total mCherry-labeled signal. Each point = 1 mouse (n=6 mixed male and female mice). Brown-Forsythe (643.8 (2.000, 6.920, p<0.0001) and Welch ANOVA (339.9 (2.000, 8.829), p<0.0001) with Dunnett’s T3 multiple comparisons test. D) Quantification of the colocalized GFAP + astrocytes with mCherry-labelled signal in the hippocampus using ImageJ shown as percentage of the total GFAP + astrocytes. 8-10 male mice sampled. E) Representative images of MFG-E8 (white) immunoreactivity in the 6-mo WT and NL-F KI SR hippocampus post control or test CRISPR-saCas9 injection. Scale bar = 20 μm. F) Quantification of MFG-E8 immunoreactivity using ImageJ. Data shown as normalized to contra control CRISPR injected hemisphere. Transparent points = individual ROIs (3 ROIs per condition; 24 ROIs sampled in total per condition), full points = mouse average of ROIs (n=8 male mice per condition). Two-way ANOVA (Interaction: F (1, 28) = 218.6, p<0.0001) followed by Bonferroni’s multiple comparisons post-hoc test on Y=log(Y) transformed mouse average. G) Representative 3D rendered images of excitatory post-synaptic Homer1 (white), CD68 lysosomes (red) and P2Y12 (green) in the 6-mo WT and NL-F KI CA1 SR hippocampus post control or test CRISPR-saCas9 injection. Scale bar = 10 μm. Inset shows representative zoom of Homer1 inside CD68 + microglial lysosomes. H) Quantification of microglial Homer1 engulfment using Imaris 3D surface rendering shown as: Homer1 volume in CD68 + lysosomes in P2Y12 + microglial surface/P2Y12 volume x100. Transparent points = individual microglia (5-6 microglia per mouse; 24 WT control, 24 NL-F KI control, 24 WT test and 34 NL-F KI test injected microglia were sampled in total), full points = mouse average of ROIs (n=4-6 male mice per condition). Two-way ANOVA (Interaction: F (1, 14) = 4.929, p<0.05) followed by Bonferroni’s multiple comparisons post-hoc test on Y=log(Y) transformed mouse average. I) Representative images of excitatory post-synaptic Homer1 (green) and pre-synaptic Bassoon (magenta) immunostaining in the 6-mo WT and NL-F KI CA1 SR hippocampus post control or test CRISPR-saCas9 injection using super-resolution Airyscan confocal microscopy. Scale bar = 1 μm. Insets show regions of colocalization. J) Quantification of the number of colocalized Homer1 and Bassoon spots shown as density per µm using Imaris. Transparent points = individual ROIs (3 ROIs per mouse; 12 WT control, 12 NL-F KI control, 12 WT test and 15 NL-F KI test injected ROIs were sampled in total), full points = mouse average of ROIs (n=4-5 male mice per condition). Two-way ANOVA (Interaction: F (1, 13) = 11.02, p<0.01) followed by Bonferroni’s multiple comparisons post-hoc test on Y=log(Y) transformed mouse average. All data shown as mean ± SEM. p-values shown ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Article Snippet: A PCR reaction was performed using 100 nM of the purified RNA product, 200 ng of the amplified gDNA region product and 1 μM EnGen® Sau Cas9 (#M0654, NEB), according to the manufacturer, and running the reaction at 37°C for 1 hr, followed by 5 min at 75°C.

Techniques: CRISPR, Knockdown, Control, Immunostaining, Construct, Injection, Labeling, Transformation Assay, Confocal Microscopy