human genome wide crispri v2 libraries  (Addgene inc)

Journal: bioRxiv

Article Title: Ribosome-associated quality control of aberrant protein production during amino acid limitation

doi: 10.64898/2026.01.14.699605

Figure Lengend Snippet: (A) A dual reporter system with a Flag-mCherry CGG -4xCGG-DHFR CGG (internal control, no translation disruption) and a Flag-YFP CGG -4xAGA-DHFR CGG was integrated into the AAVS1 locus in cells expressing CRISPRi or CRISPRa machinery (see Methods) to query the effect of a genome-wide library of CRISPRi/a sgRNAs on specific translation disruption signal. Flow cytometry was used to sort high and low responding populations. (B-D) Total score (phenotype score * Mann-Whitney (M-W) p-value, see Methods) versus hit rank for highest (high YFP) and lowest (low YFP) scoring genes in K562 CRISPRi (B), 293T CRISPRa (C), and 293T CRISPRi (D) screens. Hits are labeled and colored by associated function. Dashed line marks score cut-off where FDR = 0.25. (E) Diagram connecting major screen hit pathways. Hits are colored by direction of phenotype; blue gene labels reduce translation disruption product accumulation, red gene labels increase translation disruption product accumulation.

Article Snippet: Genome-wide CRISPRi and CRISPRa sgRNA libraries (hCRISPRi_v2: Addgene #83969 and #83970; hCRISPRa_v2: #83978 and #83979) were amplified, packaged into lentiviral particles, and titers were determined as described in ref . For K562, CRISPRi parental cells (187 million) were infected at a multiplicity of infection (MOI) of 0.28 and selected using 2 μg/mL puromycin (Sigma) for 6 days starting 48 h post-transduction.

Techniques: Control, Disruption, Expressing, Genome Wide, Flow Cytometry, MANN-WHITNEY, Labeling

Journal: bioRxiv

Article Title: Ribosome-associated quality control of aberrant protein production during amino acid limitation

doi: 10.64898/2026.01.14.699605

Figure Lengend Snippet: (A) Schematic outlining how the screen with reporters and sorting scheme depicted in was performed. Cells were arginine limited for 3 days, sorted, recovered, and re-sorted into the same bin after a second period of arginine limitation. After recovery, guide RNAs were sequenced to calculate enrichment scores. (B,C) Western blot (B) and flow cytometry (C) validation of selected hits with negative or positive phenotype scores across various pathways in K562 cells. Cells expressing CRISPRi targeting hits (or non-targeting controls; NTC) and dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ) were arginine limited for 3 days to assess translation disruption product levels (B,C) and signaling responses through mTOR, GCN2 and ZAKα (B). In (B), “*” marks non-specific band from blot stripping and reprobing. (D) Western blot to assess translation disruption and GCN2 response in 293T cells overexpressing GADD34 or an NTC by CRISPRa (scFv-sfGFP-GCN4-VP64) and dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ), with or without limitation for arginine for 5 days and treatment with 40 nM ISRIB. (E) Western blot to assess translation disruption with or without limitation for leucine or arginine for 7 days and treatment with 250 nM Torin1 in MiaPaCa cells expressing the Flag-YFP CGG -2xAGA-DHFR CGG reporter. (F) Flow cytometry to assess translation disruption product accumulation upon limitation for arginine with or without GCN2 knockdown by CRISPRi and 250 nM Torin1 treatment in K562 cells expressing the dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ). (G-H) Western blots to assess phospho-p38 and -JNK response to arginine or leucine limitation and 0.1 μg/mL anisomycin treatment with or without treatment with 5 μM SB202190 (p38 inhibitor) in wild-type and GCN2 KO 293T cells. (I) Western blot to confirm ZAKα KO in wildtype and GCN2 KO 293T cells as indicated. (J-K) Western blots to assess phospho-p38 and -JNK response to 0.1 μg/mL anisomycin treatment or UV irradiation (J) or arginine or leucine limitation (K) in wild-type and GCN2 KO, ZAKα KO, and ZAKα+GCN2 double KO 293T cells as indicated. (L) Flow cytometry to assess reporter fluorescence upon limitation for arginine for 6 days with or without treatment with 5 μM SB202190 in wild-type and GCN2 KO 293T cells expressing the Flag-YFP CGG -4xAGA-DHFR CGG (“AGA”) or Flag-YFP CGG -4xCGG-DHFR CGG (“CGG”) reporter as indicated. (M) Flow cytometry to assess reporter fluorescence over time upon limitation for arginine with or without treatment with 5 μM SB202190 in K562 cells with (sgGCN2) or without (sgNTC) GCN2 CRISPRi knockdown expressing the dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ). (N) Change in translation disruption reporter (Flag-YFP CGG -4xAGA-DHFR CGG (“AGA”) or Flag-YFP CGG -4xAGA-DHFR CGG (“CGG”)) mRNA level upon arginine limitation for 3 days with or without treatment with 5 μM SB202190 in 293T cells. (O) Change in translation disruption reporter mRNA level (Flag-YFP CGG -4xAGA-DHFR CGG ) upon arginine limitation for 3 days with (sgLAMTOR2) or without (sgNTC) CRISPRi knockdown of LAMTOR2 in 293T cells expressing the dual color translation disruption reporters (Flag-YFP CGG -4xAGA-DHFR CGG and Flag-mCherry CGG -4xCGG-DHFR CGG ), relative to cells expressing control guide (sgNTC 1). (C,L,N,O) Error bars represent standard error of the mean of 3 replicates. (B,D,E, G-K) Full-length reporter product is indicated (α-GFP antibody used to detect YFP, α-vinc = α-vinculin).

Article Snippet: Genome-wide CRISPRi and CRISPRa sgRNA libraries (hCRISPRi_v2: Addgene #83969 and #83970; hCRISPRa_v2: #83978 and #83979) were amplified, packaged into lentiviral particles, and titers were determined as described in ref . For K562, CRISPRi parental cells (187 million) were infected at a multiplicity of infection (MOI) of 0.28 and selected using 2 μg/mL puromycin (Sigma) for 6 days starting 48 h post-transduction.

Techniques: Western Blot, Flow Cytometry, Biomarker Discovery, Expressing, Disruption, Stripping, Knockdown, Irradiation, Fluorescence, Control

Journal: bioRxiv

Article Title: Mapping kinase-dependent tumor immune adaptation with multiplexed single-cell CRISPR screens

doi: 10.64898/2026.01.08.698516

Figure Lengend Snippet: (a) Experimental schematic of CRISPRi/a perturbations in NY-ESO-1/HLA-A*02:01-engineered GBM cells co-cultured with T cells. (b) UMAP embeddings of cancer cells under CRISPRi (left) or CRISPRa (right) showing transcriptomic trajectories across E:T ratios. (c) Module-level changes in MHC-I, MHC-II, IFN-γ, and NF-κB pathway programs under CRISPRi versus CRISPRa. Colors indicate effect size, quantified as the β coefficient for the genotype term (perturbation) in a regression model evaluated under the no–T cell condition. (d) Schematic for MrVI analysis, and (e) Forest plots summarizing perturbation effects on T cell-induced program across E:T ratios from MrVI analysis. Color and circle size denote p values, while arrow length and thickness represent distance differences. The statistical tests were performed with a one-sided Mann-Whitney U test (Bonferroni-corrected, α < 0.05).

Article Snippet: Protospacer sequences targeting all genes perturbed in this study were sourced from the genome-wide human CRISPRi and CRISPRa v2 libraries developed by Horlbeck et al. (CROP-seq-OPTI, Addgene, Plasmid #106280, engineered to include a CRISPRi-optimized gRNA backbone) were synthesized by Integrated DNA Technologies (IDT).

Techniques: Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: Mapping kinase-dependent tumor immune adaptation with multiplexed single-cell CRISPR screens

doi: 10.64898/2026.01.08.698516

Figure Lengend Snippet: (a) Decipher analysis of NTC cells from CRISPRa data reveals trajectories driven by increasing T cell concentration, shown in the learned 2D Decipher space with cells colored by E:T ratios (left) and corresponding Decipher time (pseudotime; right). (b) Genes significantly contribute to the Decipher trajectory, with the positions of IDO1 and SOD2 highlighted. (c) Single-cell trajectories of representative genes inferred by Decipher reveal E:T ratio-dependent transcriptomic responses. (d) Decipher-aligned CRISPRi and CRISPRa cells (left) projected into a shared 2D Decipher space together with NTC cells (right). (e) Decipher trajectories learned as a function of E:T ratio (left) and pseudotime (right) for CRISPRi and CRISPRa conditions. (f) Comparison of CRISPRi and CRISPRa perturbations highlights conserved effects on immune checkpoint expression ( CD274 ) and tumor-intrinsic T cell-responsive genes ( CSF3 ). (g) Density plots illustrate distribution shifts of cancer cells along Decipher time following perturbation of selected kinases ( EPHA2 , STK40 ), indicating that kinase disruption alters the progression of cancer cell states under T cell pressure (p = 0.033639216, p = 0.008503096 with Kolmogorov-Smirnov test).

Article Snippet: Protospacer sequences targeting all genes perturbed in this study were sourced from the genome-wide human CRISPRi and CRISPRa v2 libraries developed by Horlbeck et al. (CROP-seq-OPTI, Addgene, Plasmid #106280, engineered to include a CRISPRi-optimized gRNA backbone) were synthesized by Integrated DNA Technologies (IDT).

Techniques: Concentration Assay, Comparison, Expressing, Disruption