Journal: bioRxiv

Article Title: High-throughput CRISPR live-cell imaging of low-frequency chromosomal events quantifies the latent efficiency of chromosome engineering

doi: 10.64898/2026.05.10.724155

Figure Lengend Snippet: (A) Schematic of the recombinant dCas9 used for chromosome labeling in live cells. Two green fluorophores (sfGFP and mNeonGreen) are fused at the N terminus, and three tandem 2× nuclear localization signal (NLS) modules are positioned at the N terminus, an internal site, and the C terminus to enhance nuclear import. (B) Representative labeling of a mouse artificial chromosome (MAC) in an HT1080 MAC-positive line imaged by confocal microscopy. Nuclei are stained with Hoechst 33342. The inset highlights a representative CRISPR spot (arrow). Scale bars, 50 µm (overview) and 5 µm (inset). (C) Per-cell signal-to-noise ratio (SNR) distribution for MAC-positive and MAC-negative HT1080 cell lines (n = 262 MAC-positive cells and n = 160 MAC-negative cells across three independent experiments). Per-cell SNR was defined as the maximum intranuclear spot SNR within each nucleus. For cells with no detectable spot, the SNR was set to 0. (D) Precision–recall (PR) performance for MAC detection obtained by sweeping the SNR threshold. The curve shows mean performance across three independent experiments; the shaded band indicates ± standard deviation (s.d.); mAP is mean average precision. (E) Two-color colocalization assay using dCas9–green (sfGFP–mNeonGreen) and dCas9– red (2×mScarlet) programmed with distinct guide sets targeting the MAC (sgMajSat_1 for green and sgMajSat_2 for red). Nuclei are stained using NucSpot Live 650. The inset highlights a representative CRISPR spot of MAC focus (arrow). Colocalization was assessed by intersection-over-union (IoU) between the green and red focus masks, defined as the area of overlap divided by the area of union; spots were classified as colocalized at IoU ≥ 0.5. Scale bars, 5 μm.

Article Snippet: For the MAC-positive HT1080 cell line (HT1080 MI-MAC27-2), G418 (InvivoGen, #ant-gn-1) was added to the medium at 600 μg/ml.

Techniques: Recombinant, Labeling, Confocal Microscopy, Staining, CRISPR, Standard Deviation

Journal: bioRxiv

Article Title: High-throughput CRISPR live-cell imaging of low-frequency chromosomal events quantifies the latent efficiency of chromosome engineering

doi: 10.64898/2026.05.10.724155

Figure Lengend Snippet: (A)Schematic of workflow modifications to reduce nonspecific puncta: protease treatment to remove membrane-associated RNP aggregates and transient glutamine deprivation to reduce intranuclear nonspecific accumulation. (B)Representative z-projection image of an OPM volume acquired from MAC-positive HT1080 cells (acquisition time, ~1.5 s per volume). Nuclei and CRISPR spots are shown in magenta and green, respectively. Scale bar, 100 μm. (C)Representative intranuclear CRISPR spots. Maximum-intensity z-projections of the GFP channel are shown; nuclear boundaries are outlined in yellow and arrows indicate discrete spots. Scale bar, 5 µm. (D)Cell-level SNR distributions without and with the workflow optimizations. Vertical lines denote the SNR threshold used for calling and the corresponding limit of detection (LoD) under the defined criteria. Blue, positive-control cells (n = 16,831 without optimization; n = 13,056 with optimization); orange, negative-control cells (n = 20,127 without optimization; n = 16,994 with optimization). (E)Precision–recall curves without and with the workflow optimizations by sweeping the SNR threshold on datasets from 30,723 MAC-positive cells and 37,577 MAC-negative cells across three independent experiments. mAP, mean average precision (without optimization, 0.941; with optimization, 0.971) and standard deviation (shadow).

Article Snippet: For the MAC-positive HT1080 cell line (HT1080 MI-MAC27-2), G418 (InvivoGen, #ant-gn-1) was added to the medium at 600 μg/ml.

Techniques: Membrane, CRISPR, Positive Control, Negative Control, Standard Deviation

Journal: bioRxiv

Article Title: High-throughput CRISPR live-cell imaging of low-frequency chromosomal events quantifies the latent efficiency of chromosome engineering

doi: 10.64898/2026.05.10.724155

Figure Lengend Snippet: (A) Experimental schematic. Following microcell fusion and expansion, recipient HT1080 cells were split into two arms: Hi-CRI imaging after 1 day of culture, or antibiotic selection followed by a clonogenic assay after 8 days. (B) Representative z-projection from an OPM volume in the post-MMCT population. Nuclei (magenta) and CRISPR spots (green) are shown. The inset shows a MAC-positive cell, in which the nuclear boundary is outlined in yellow and the arrow marks an intranuclear CRISPR spot. Scale bars, 75 µm (overview) and 3 µm (inset). (C) Estimated MAC-positive fraction measured by Hi-CRI imaging and by antibiotic-selection-based clonogenic assay across three independent MMCT experiments (Rep 1–3; y-axis, log scale). For Hi-CRI, the fraction is the number of cells called positive divided by the number of analyzed cells; for the clonogenic assay, the fraction is the number of surviving colonies divided by the number of input cells. Dots indicate technical replicates, ranging from 14,097 to 33,032 cells per replicate; error bars indicate ± s.d.

Article Snippet: For the MAC-positive HT1080 cell line (HT1080 MI-MAC27-2), G418 (InvivoGen, #ant-gn-1) was added to the medium at 600 μg/ml.

Techniques: Imaging, Selection, Clonogenic Assay, CRISPR