crispr cas9 cleavage site  (Integrated DNA Technologies)

Journal: The Journal of Experimental Medicine

Article Title: Multi-omic comparison of Alzheimer’s variants in human ESC–derived microglia reveals convergence at APOE

doi: 10.1084/jem.20200474

Figure Lengend Snippet: Integrating AD-associated genetic variations into an hMGL model. (A) Schematic depiction of AD-associated risk variants characterized. SNP variants for CD33 and INPP5D , as well as R47H, A528T, and R744X coding variants for TREM2 and SORL1 (SORLA) are marked in red. ITIM, immunoreceptor tyrosine-based inhibitory motif. (B) Workflow pipeline, for generating and characterizing AD-associated mutations in hMGLs. AD-associated coding or noncoding SNPs are introduced into corresponding genomic loci in human H9 ESC lines by CRISPR-Cas9 editing. Each line was characterized for targeted mutations and off-targeting variation before differentiation and maturation into hMGLs. hMGLs were subjected to multi-omic (RNA-seq, ATAC-seq, ChIP-seq, and label-free proteome) analysis, and functional characterization as indicated. (C) Isogenic microglial differentiation scheme used in this study. ESCs were differentiated into HPCs for 10 d, where CD43 + iHPCs are sorted (FACS plots) and cultured in serum-free media with MCSF, IL-34, TGF-β, and insulin; CD43 (green), CX3CR1 (red), Iba1 (purple), and DAPI (blue) staining is shown for HPCs at 10 d in vitro (DIV). Cells were differentiated to microglia for an additional 25 d, whereby maturation was induced by the addition of CD200 and CX3CL1. hMGLs were stained for TREM2 (red), CD43 (green), Iba1 (purple), and DAPI (blue) and compared with HPCs (bottom panels), or TMEM119 in hMGLs (red, bottom right) as indicated. Scale bars represent 100 µm (H9, left panel), 50 µm (mature hMGLs, right panel), and 20 µm (all fluorescence images). (D) Heatmap depicting RNA-seq profiles from human microglia (red; ; GSE99074 , red), hMGLs from this study (purple), iMGLs ( ; GSE117829 , green). (E) 3D PCA of hMGLs (this study, purple), iMGLs ( GSE117829 , turquoise; GSE89189 , dark blue), human fetal microglia ( GSE89189 , green), human adult microglia ( GSE89189 , light blue), myeloid dendritic cells ( GSE89189 , light yellow), monocytes ( GSE89189 , gold). PCA reveals that hMGLs cluster closely with iMGLs and human adult/fetal microglia, and are distinct from myeloid cells.

Article Snippet: 100-nt single-stranded oligodeoxynucleotide (ssODN) repair templates (PAGE purified; Integrated DNA Technology) were designed with homologous genomic sequences flanking the predicted CRISPR-Cas9 cleavage site ( ).

Techniques: CRISPR, RNA Sequencing Assay, ChIP-sequencing, Functional Assay, Cell Culture, Staining, In Vitro, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Multi-omic comparison of Alzheimer’s variants in human ESC–derived microglia reveals convergence at APOE

doi: 10.1084/jem.20200474

Figure Lengend Snippet: Gene targeting and experimental strategy for hMGL differentiation and characterization. (A) Schematic representation of the genomic location and intron/exon schematic of AD risk SNPs CD33 , INPP5D , TREM2 , and SORL1 in this study. (B) Schematic diagram of the analytical workflow for this study. RNA-seq datasets from the hMGL lines (1) are analyzed for cross-regulatory interactions to generate an epistatic model (2) and identify potential pathogenic effectors or signatures. hMGL lines are characterized for physiological microglial function (3) and interactions with Aβ in immunodeficient human MCSF knockin mouse brain xenotransplants (4). (C) Representative sequences of various isogenic clones in AD-associated mutant ESC lines and H9-WT sequences. Repair single-strand donor (ssODN) templates, sgRNA (gray), corresponding amino acids, DNA directionality (arrow, 5′ to 3′) and nucleotide substitutions are shown. For TREM2 R47H , two synonymous mutations were introduced in the repair ssODN, generating a new HindIII restriction site (lowercase) for consequent clone screening. (D) Sanger sequencing and validation of CD33 SNP, INPP5D SNP, TREM2 KO, TREM2 R47H , SORL1 KO, and SORL1 A528T lines and isogenic controls (nontargeting sgRNA). The WT H9 ESC line is heterozygous for G/A INPP5D SNPs; CRISPR-Cas9 editing was performed to convert H9 homozygously to the INPP5D “A” allele. All other modifications were converted homozygously in the H9 ESC lines. (E) After maturation induced by exposure to CD200 and CX3CL1, hMGLs were stained for CX3CR1 (red), CD43 (green), Iba1 (purple), and DAPI (blue) as indicated. Scale bar, 20 µm. (F) Representative inward currents from WT hMGLs; hyperpolarizing voltage steps from −160 mV to −60 mV were applied in the absence (top) or presence of Cs + (bottom). At right panel, quantification of inward currents as measured in the absence (black) or presence of Cs + (gray). (G) Induction of cytokines and chemokines in WT hMGLs stimulated with IL-1β (20 ng/ml) and IFN-γ (20 ng/ml) as determined by ELISA multiplex assay. Heatmaps indicate log 2 fold change of cytokines/chemokines indicated (MCP-1, GPOa, HGF, TNFα) above vehicle treatment. Results are from three replicate cultures in three independent experiments. (H) Representative time-lapse images showing WT hMGL migration toward to ATP source (a pipette tip). (I) Representative images of calcium imaging over the time periods as indicated with 100 µM ATP stimulation. Scale bar, 25 µm. Graphs (right) depict Ca 2+ traces depicting changes in Fluo-4 fluorescence over the baseline (ΔF/F0) in response to 100 µM ATP in the WT hMGLs. Results are derived from averaged values in three replicate cultures and three experiments. (J) Representative time-lapse images of fluorescent Aβ 1-42 oligomers (red) bound to WT hMGLs, imaged by automated live-cell microscopy. In the adjacent graph, phagocytosis of Aβ 1-42 oligomers in WT hMGLs over time was quantified, as depicted on the left. PI was determined by measuring average fluorescence intensity at each time point in comparison to the 15-min time point (set to 1.0). Images in E–J are representative of three independent experiments. Values represent mean ± SEM from n = 3 independent experiments.

Article Snippet: 100-nt single-stranded oligodeoxynucleotide (ssODN) repair templates (PAGE purified; Integrated DNA Technology) were designed with homologous genomic sequences flanking the predicted CRISPR-Cas9 cleavage site ( ).

Techniques: RNA Sequencing Assay, Knock-In, Clone Assay, Mutagenesis, Sequencing, CRISPR, Staining, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Migration, Transferring, Imaging, Fluorescence, Derivative Assay, Microscopy