Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , b , Schematic of genome-wide CRISPR off-target detection using MRE11 ChIP–seq. a , Cells are unsynchronized, so only some cells have MRE11 at Cas9 cut sites at a given time (DISCOVER-Seq). b , Inhibition of NHEJ directs DNA repair to slower, MRE11-dependent pathways. c , Effect of repair factor inhibition on MRE11 residence at the VEGFA site 3 on-target site, measured by ChIP–qPCR estimating ‘reads per million’ (RPM) enrichment at 12 h after Cas9 delivery in HEK293T cells. Each point corresponds to a different biologically independent replicate of a sample exposed to the DNA repair inhibitor listed in the x axis. Red line is the mean of two biologically independent replicates. Samples with DNA-PKcs inhibition ( n = 4) have significantly higher estimated RPM compared with samples without DNA-PKcs inhibition ( n = 8) using two-sided Student’s t -test ( P = 9.65 × 10 −5 ). d , Increased MRE11 residence upon DNA-PKcs inhibition using Ku-60648 (red) versus without inhibition (blue), measured by ChIP–qPCR. Measured over multiple time points (4 h, 12 h, 24 h) after delivery of Cas9 targeting VEGFA site 2 in HEK293T (left plot), with Cas9 targeting FANCF site 2 in K562 at 12 h (middle plot) and with Cas9 targeting HEK site 4 in HEK293T at 12 h (right plot). Plots display the mean over two biologically independent replicates for left and middle plots, and one biologically independent replicate for the right plot. e , Plot of estimated RPM enrichment normalized to the no drug sample from data in panel d , for sample pairs with (‘Ku-60648’) or without (‘no drug’) DNA-PKcs inhibition. Normalized RPM enrichment with DNA-PKcs inhibition was significantly higher than without inhibitor ( P = 0.0001), using two-sided Wilcoxon signed-rank test. Red line indicates mean of n = 14 total samples pooled from panel d ; green points are HEK293T, VEGFA site 2 ; purple points are HEK293T, HEK site 4 ; red points are K562, FANCF site 2 . *** P < 0.001.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: Genome Wide, CRISPR, ChIP-sequencing, Inhibition, ChIP-qPCR

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , The proportion of 53BP1 foci relative to BRCA1 as detected by STED in cells exposed to Cas9 targeting a multi-target gRNA with 126 genome-wide target sites. N = 98 cells examined over four independent experiments, P = 0.00018 using two-sided Wilcoxon rank-sum test. b , The number of repair foci (53BP1 or BRCA1) as detected by STED in cells with or without Cas9 (‘+Cas9’ or ‘−Cas9’, respectively), with or without Ku-60648 (‘KU’ versus ‘nD’, respectively), targeting 126 genome-wide sites with a multi-target gRNA. N = 98 cells from four biologically independent replicates, P = 4.44 × 10 −10 using two-sided Wilcoxon rank-sum test between ‘−Cas9’ and ‘+Cas9’. Difference in no. of foci in each group was not significant (left, P = 0.15; right, P = 0.95). c , d , Representative images for panel b , ( c ) with Cas9 or ( d ) without Cas9. Red labels 53BP1, green labels BRCA1. Scale bar, 5 μm. e , Histogram of indels or no mutations (‘0’) at 48 h after Cas9 editing of ACTB , either without DNA-PKcs inhibitor (‘Cas9, no inhibitor’) or with inhibitor (‘Cas9, Ku-60648’). Untreated cells not exposed to Cas9 shown for reference (‘Untreated (no Cas9)’). Red bar displays mean over two biologically independent replicates. *** P < 0.001. NS, not significant.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: Genome Wide

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , b , Plots of MRE11 ChIP–seq enrichment (number of reads within a 1.5-kb window centered at the cut site, per 1 million total reads, that is, RPM) for samples with ( y axis) or without ( x axis) DNA-PKcs inhibition at all FANCF site 2 ( a ) or VEGFA site 2 ( b ) Cas9 target sites in K562 detected from the DNA-PKcs inhibited samples. Each point in the plot (15 in panel a , 178 in panel b ) corresponds to a putative target site. Significant differences ( P = 0.00081 or P = 4.57 × 10 −26 ) between y -axis and x -axis values were determined using two-sided Wilcoxon signed-rank test. c , d , Genome browser visualization of MRE11 enrichment at an on-target ( c ) and representative off-target ( d ) position from K562 with Cas9 targeting FANCF site 2 , with (red) or without (blue) DNA-PKcs inhibition. e , f , Plots of MRE11 ChIP–seq enrichment for samples with ( y axis) or without ( x axis) DNA-PKcs inhibition at positions 10 kb downstream from the actual ( e ) FANCF site 2 or ( f ) VEGFA site 2 cut sites, to measure background enrichment adjacent to cut sites. MRE11 enrichment with ( y axis) versus without ( x axis) DNA-PKcs inhibition at the adjacent background locations was not significantly different ( P = 0.21 or 0.18), determined using two-sided Wilcoxon signed-rank test. g , Number of discovered off-target sites with (red) or without (blue) DNA-PKcs inhibition for VEGFA site 2 . Quantification of Extended Data Fig. . h , i , Number of discovered off-target sites with (red) or without (blue) DNA-PKcs inhibition for VEGFA site 3, FANCF site 2 ( h ) and HEK site 4 ( i ) gRNAs. Quantification of Extended Data Fig. . j , Venn diagram illustrating overlap in the identity of Cas9 target sites discovered from samples with DNA-PKcs inhibition (‘DNA-PKi only’; light blue), without DNA-PKcs inhibition (‘no drug only’; light yellow) or found in both samples (‘both’; light green). Four gRNAs were evaluated.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: ChIP-sequencing, Inhibition

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , Illustration of MRE11 ChIP-seq enrichment at a specific target site, visualized as a histogram of base pair coverage from sequencing reads along the genome. Only the two ends of each DNA fragment are sequenced. b , Genome browser visualization of MRE11 enrichment at the on-target site in K562 cells with Cas9 targeting FANCF site 2 . Y-axis is in log 10 scale. Overlaying red and blue graphs correspond to with or without DNA-PKcs, respectively. Black graph corresponds to samples without Cas9.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: ChIP-sequencing, Sequencing

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , VEGFA site 2 Cas9 target sites detected using DISCOVER-Seq (left) versus DISCOVER-Seq+ (right) in K562 cells. b , FANCF site 2, VEGFA site 3, or HEK site 4 Cas9 target sites detected using DISCOVER-Seq versus DISCOVER-Seq+ in K562 or HEK293T cells, at 4 h, 12 h, or 24 h after Cas9 delivery. Source numerical data are available in source data.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques:

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a-b , Plot of the total number of initial off-target sites, for samples (a) without inhibitor and (b) with Ku-60648. Sites labeled as false positive, because they were also reported in corresponding negative control samples without Cas9, are colored red with the number of sites indicated above each bar. For x-axis, first row is cell type, second row is gRNA target (Vs2: VEGFA site 2, Hs4: HEK site 4, Vs3: VEGFA site 3, Fs2: FANCF site 2). c , Plot of MRE11 ChIP-seq enrichment at all DISCOVER-Seq+ detected target sites within a 1.5 kb window for Cas9 targeting VEGFA site 2 in WTC-11 iPSCs. Each point in the plot (55 total) corresponds to a putative target site. MRE11 enrichment from DISCOVER-Seq+ data (y-axis) versus DISCOVER-Seq data (x-axis) was significantly different (p = 1.24E-8), determined using the two-sided Wilcoxon signed-rank test. d-e , Venn diagram illustrating overlap in the VEGFA site 2 sites identified by (d) DISCOVER-Seq or (e) DISCOVER-Seq+ in K562 cells (blue), HEK293T cells (yellow), and WTC-11 iPSCs (purple). f-g , (f) Flow cytometry contour plots of T cell populations at day 7 following CRISPR editing with Cas9 (left) or Cas12a (right). X-axis gates by T cell binding to a phycoerythrin (PE)-conjugated HLA-A*02:p53 R175H peptide tetramer complex specific for the tgTCR. Y-axis gates by allophycocyanin (APC)-conjugated antibody against NGFR (CD271), introduced as part of the CRISPR HDRT as an editing control. Cells positive for both markers represent successful cancer-specific tgTCR integration. 9.7% of T cells are positive for both markers using Cas9 compared to 8.4% with Cas12a. (g) Same, but for negative control samples without gRNA. h , Gating strategy for flow cytometric analysis of live single T cells, used for panels f-g . i , Same as panel c , for Cas9 targeting PCSK9 in the liver of mice, with 30 total target sites. MRE11 enrichment from DISCOVER-Seq+ was significantly different (p = 7.90E-6), using the two-sided Wilcoxon signed-rank test. Source numerical data are available in source data.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: Labeling, Negative Control, ChIP-sequencing, Flow Cytometry, CRISPR, Binding Assay, Control

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , For the 15 target sites (1 on-target, 14 off-targets) of the FANCF site 2 gRNA identified by DISCOVER-Seq+ (‘DSeq+’), the chart shows which sites are also identified by DISCOVER-Seq alone (‘+’ under ‘DSeq’), which sites have indels detectable by targeted deep sequencing (‘+’ under ‘Indels’) and which sites were also detectable by GUIDE-seq (‘+’ under ‘Gseq’). Target sites labeled with ‘N/A’ under ‘Indels’ were unable to be successfully amplified by PCR for targeted sequencing. b , Left plot, measurement of indels at the on-target and sole off-target site (OFF0) discovered by the original DISCOVER-Seq, for K562 cells with the FANCF site 2 gRNA, with or without Cas9 (‘+Cas9’ or ‘−Cas9’, respectively). Right plot, measurement of indels at off-target sites exclusively discovered by DISCOVER-Seq+. Plots display the mean of three biologically independent replicates; error bars represent ±1 s.d. from mean. * P < 0.05, ** P < 0.01 and *** P < 0.001, using two-sided Student’s t -test (exact P values in the for this figure). c , Venn diagram illustrating overlap in the identity of VEGFA site 2 and FANCF site 2 target sites identified by DISCOVER-Seq+ versus GUIDE-seq.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: Sequencing, Labeling, Amplification

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , VEGFA site 2 Cas9 target sites detected using DISCOVER-Seq (left) versus DISCOVER-Seq+ (right) in WTC-11 iPSCs. b , c , Genome browser visualization of MRE11 enrichment at an on-target ( b ) and representative off-target ( c ) position with four mismatches (‘4 mm’) in WTC-11 iPSCs with Cas9 targeting VEGFA site 2 . DISCOVER-Seq+ data in red (with Ku-60648), DISCOVER-Seq data in blue (with no drug exposure). d , Schematic of the DISCOVER-Seq+ protocol in the knock-in of a cancer neoantigen-specific tgTCR into the TRA locus of primary human T cells. e , TRA Cas9 target sites in primary T cells detected using DISCOVER-Seq (left) versus DISCOVER-Seq+ (right). f , Genome browser visualization of MRE11 enrichment at a representative four-mismatch (‘4 mm’) off-target position in primary human T cells with Cas9 targeting TRA for knock-in of a tgTCR template. DISCOVER-Seq+ data in red (with Ku-60648), DISCOVER-Seq data in blue (with no drug exposure). g , Same as panel f , at another four-mismatch off-target position. h , Plot of MRE11 ChIP–seq RPM enrichment within a 1.5-kb window for samples with ( y axis) or without ( x axis) DNA-PKcs inhibition, at all TRA Cas9 off-target sites in primary human T cells from the DNA-PKcs inhibited samples. Each point in the plot (20 total) corresponds to a putative target site. Differences ( P = 1 × 10 −3 or P = 0.69) between y -axis and x -axis values were determined using two-sided Wilcoxon signed-rank test. i , Same as panel h , for cells delivered with Cas9 but without gRNA (negative control). j , TRA Cas12a (Cpf1) target sites in primary T cells.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: Knock-In, ChIP-sequencing, Inhibition, Negative Control

Journal: Nature Methods

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1038/s41592-023-01840-z

Figure Lengend Snippet: a , Schematic of DISCOVER-Seq+ protocol in mice. b – d , Genome browser visualization of MRE11 enrichment at the ( b ) PCSK9 on-target site (‘ON-target’), ( c ) one off-target site (‘OFF-target A’) and ( d ) another off-target site (‘OFF-target B’) with two mismatches each (‘2 mm’), in the liver of mice transduced with adenovirus expressing Cas9 targeting PCSK9 . Mice were dosed twice a day (b.i.d.) with either 25 mg kg −1 Ku-60648 (‘Ku-60648’; red) or with vehicle (‘no drug’; blue). e , Number of detected genome-wide target sites in the mouse genome mm10 with Cas9/gRNA targeting PCSK9 , identified using DISCOVER-Seq (DSeq) versus DISCOVER-Seq+ (DSeq+). N = 5 biologically independent replicates (mice) were used for each condition; two-sided Student’s t -test was used ( P = 0.0079). f , Venn diagram illustrating overlap in the mouse PCSK9 target sites identified by in vivo DISCOVER-Seq+ in this work, ‘DSeq+ (this work)’ (blue); by in vivo DISCOVER-Seq in this work, ‘DSeq (this work)’ (yellow); and in the original DISCOVER-Seq manuscript, ‘DSeq (Wienert et al. )’ (purple). All the target sites identified by ‘DSeq (this work)’ are also found in the other two groups. g , PCSK9 Cas9 target sites detected using DISCOVER-Seq (left) versus DISCOVER-Seq+ (right) in mouse liver. Off-target detection for each condition was performed on sequencing data pooled across five biologically independent mouse replicates. ** P < 0.01.

Article Snippet: Cas9 nuclease V3 (IDT) or Alt-R A.s. Cas12a (Cpf1) Ultra nuclease (IDT) and matching ssDNA Electroporation Enhancer (IDT) and incubating the mixture at room temperature for 15 min. RNPs were mixed with 0.5 μg of the same HDRT and incubated for 5 min at room temperature.

Techniques: Transduction, Expressing, Genome Wide, In Vivo, Sequencing

Journal: bioRxiv

Article Title: Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier

doi: 10.1101/2025.09.23.678106

Figure Lengend Snippet: a Representative fluorescent image of BBB organoids formed from EphA2 knockout (KO) human brain endothelial cells. EphA2 KO cells express RFP (magenta) against DAPI (blue) counterstaining. b Western blot analysis (1:1000 dilution of rabbit anti-Human EphA2; Cell signaling technology Inc. D4A2) (1:10,000 dilution of Goat Anti-Rabbit IgG H&L HRPab6721; Abcam Inc., Cambridge, MA, USA) confirming loss of EphA2 protein expression in EphA2 KO organoids, in contrast to controls (EphA2+ and EphA2 over-expression (o/e)). c Internalization of Cn ( Cryptococcus neoformans ) by BBB organoids is dependent on EphA2 expression. Organoids (EphA2+ or EphA2 KO brain endothelial cells) were exposed to CFSE-stained Cn for 48 h, after which organoids were sectioned and analyzed for Cn invasion (Welch two-sample t-test: t = 4.28, df = 13.94, p < 0.0008)(R v.4.4.2). d Comparison of GFAP expression between BBB organoids formed with EphA2 KO versus EphA2+ brain endothelial cells. Organoids deficient in EphA2 show significantly higher levels of GFAP expression compared to EphA2+ organoids. Quantification was performed on GFAP-antibody probed organoid sections by applying a uniform threshold to images and measuring the percent area in the relevant channel above the threshold (Welch two-sample t-test: t = 2.42, df = 10.68, p = 0.0343)(R v.4.2.2). e Organoids exposed to Cd ( Cryptococcus deuterogattii ) show increased GFAP expression in stark contrast to organoids exposed to Cn after 48 h of exposure (Welch two-sample t-test comparing Cn & Cd treatments: t = 3.84, df = 9.38, p = 0.003, comparing Cd & Control treatments: t = 4.25, df = 10.57, p = 0.0015)(R v.4.4.2). For panels c – e, each data point represents the average value for an organoid across several images.

Article Snippet: Two sets of plasmids were used in the CRISPR/Cas9 method - the EphA2 CRISPR/Cas9 KO plasmids and the EphA2 HDR plasmids, both of which were ordered from Santa Cruz Biotechnology.

Techniques: Knock-Out, Western Blot, Expressing, Over Expression, Staining, Comparison, Control

Journal: bioRxiv

Article Title: Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier

doi: 10.1101/2025.09.23.678106

Figure Lengend Snippet: a Cn induced stimulation of GTP-bound Cdc42 is contingent on EphA2 expression and b CD44 engagement. EphA2+ and EphA2 KO human brain endothelial cells were treated with either a Cn or b cps1Δ Cn deletion mutant at an MOI of 10 for 30 min and then lysed. Untreated and treated cell lysates were analyzed for GTP bound Cdc42 (active Cdc42) using the Cytoskeleton G-LISA assay. Data are expressed as a mean ± SD of three independent experiments. Statistical analysis was done using a two tailed unpaired t-test (n = 8, t = 0.2156, df = 14, **** p < 0.0001)(GraphPad Prism 10 software). c, d Expression of Cdc42 protein in lysates was confirmed by western blot analysis of nitrocellulose membrane probed with primary antibody to amino acid 150-182 of Cdc42 protein. (1:800 dilution of mouse anti-human Cdc42; cytoskeleton Inc. ACD04) (1:10,000 dilution of Goat anti-Mouse IgG H&L HRP ab6789; Abcam Inc., Cambridge, MA, USA). e, f Cn recruits a CD44-EphA2 protein complex in brain endothelial cells, detected by Proximity Labeling Assays (PLA - DuoLink) of EphA2 and CD44 in e mouse primary brain endothelial cells and f human brain endothelial cells (EphA2+, iBMEC). The in-situ interaction was quantified as the area in an image above a universally applied threshold. Significantly higher fluorescence was observed in Cn (+) versus Cn (-) treatments in both mouse primary cells (t = 4.40, df = 43.13, p < 0.0001) and human cells (t = 3.63, df = 131.88, p = 0.0004). In mouse primary cells, there was significantly higher signal in the Cn (+) compared to the Cd (+) treatments (t = 4.08, df = 43.63, p < 0.0002)(R v.4.4.2). Mouse primary brain endothelial cells were exposed to Cd , Cn media or a no primary antibody control. g Representative fluorescent images of PLA in human brain endothelial cells (EphA2+, iBMECs). Top row: iBMECs exposed to Cn for 90 min. Middle row: control panel iBMECs not exposed to Cn . Bottom row: control treatment in which iBMECs were exposed to Cn but omitting primary antibodies (anti-CD44 and anti-EphA2) Left column: Nuclear stain. Middle column: Fluorescent puncta (PLA-red probe) are indicative of target proteins (EphA2 and CD44) in proximity (within 40 nm) to each other. Right column: merged images.

Article Snippet: Two sets of plasmids were used in the CRISPR/Cas9 method - the EphA2 CRISPR/Cas9 KO plasmids and the EphA2 HDR plasmids, both of which were ordered from Santa Cruz Biotechnology.

Techniques: Expressing, Mutagenesis, Two Tailed Test, Software, Western Blot, Membrane, Labeling, In Situ, Fluorescence, Control, Staining

Journal: bioRxiv

Article Title: Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier

doi: 10.1101/2025.09.23.678106

Figure Lengend Snippet: Alpha-Fold-Multimer structure prediction indicates reliable binding between EphA2 and CD44 . a Cartoon representation of the AlphaFold3-predicted model of the EphA2 structure. extracellular region in complex with CD44, highlighting two distinct putative binding interfaces: the ligand-binding domain (LBD) (ipTM = 0.13; pTM = 0.49) and the fibronectin domains (FN1-FN2) (ipTM = 0.11; pTM = 0.49). b-c Representative binding conformations of the LBD: CD44 and FN: CD44 complexes obtained from molecular dynamics simulations. Shown here are the major conformational clusters derived from all replica trajectories. EphA2 is depicted in green and CD44 in red cartoon representations. For clarity, only the principle interfacial hydrogen bonds are displayed.

Article Snippet: Two sets of plasmids were used in the CRISPR/Cas9 method - the EphA2 CRISPR/Cas9 KO plasmids and the EphA2 HDR plasmids, both of which were ordered from Santa Cruz Biotechnology.

Techniques: Binding Assay, Ligand Binding Assay, Derivative Assay

Journal: bioRxiv

Article Title: Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier

doi: 10.1101/2025.09.23.678106

Figure Lengend Snippet: Upon binding to CD44, Cn promotes the association of CD44 with the ligand binding domain (LBD) and interactions involving both FN1 and FN2 domains, suggesting that CD44 can also engage EphA2 via a membrane-proximal site and/or the fibronectin 2 domain (FN2) (black arrows). The CD44-EphA2 molecular interaction leads to a PKC-dependent phosphorylation of EphA2, and the downstream activation of GTP-bound Cdc42 (green arrows). Active Cdc42 stimulates membrane ruffling by remodeling the cytoskeleton via actin polymerization. The ensuing macropinocytosis of Cn is a result of macropinosomes large enough to internalize and transport Cn across the brain endothelium. Cd does not cross the brain endothelium (crossed-out black arrow), but stimulates the expression of GFAP+ astrocytes by as yet an unknown mechanism (dashed arrow). Figure created in BioRender.com.

Article Snippet: Two sets of plasmids were used in the CRISPR/Cas9 method - the EphA2 CRISPR/Cas9 KO plasmids and the EphA2 HDR plasmids, both of which were ordered from Santa Cruz Biotechnology.

Techniques: Binding Assay, Ligand Binding Assay, Membrane, Phospho-proteomics, Activation Assay, Expressing