Journal: Life Medicine

Article Title: A CRISPR/Cas9-based kinome screen identifies ErbB signaling as a new regulator of human naïve pluripotency and totipotency

doi: 10.1093/lifemedi/lnad037

Figure Lengend Snippet: Figure 1. CRISPR kinome KO screen identified novel targets that increase human EPS cells’ competence of acquiring authentic human naïve state. (A) Schematics of CRISPR knockout screen with kinome library under naïve or extended pluripotent conditions. (B) Plasmid design of iCas9 expression with BCL2 and Neomycin resistance. iCas9, induced Cas9. (C) Quantitative polymerase chain reaction (q-PCR) analysis of the expression of iCas9 in hEPSC-iCas9-hBCL2-Neo cell line upon Dox treatment. N = 2 biological replicates. Error bar indicates standard error of the mean (SEM). (D) The distribution diagram of the initial sgRNA library read counts frequency. (E) Representative flow cytometry sorting of SUSD2 positive cells during kinome KO screen under XF-LCDM condition. Control hEPSCs without SUSD2-phycoerythin (PE) staining (left), control hEPSCs with SUSD2-PE staining (middle), and kinome KO EPSCs with SUSD2-PE staining (right) are presented. The percentage of SUSD2-PE positive cells is shown. This experiment was repeated at least three times. (F) Kinases targeted by positively enriched sgRNAs in cells at passage 6 under naïve pluripotent condition 5i/LA (left) and PXGL (right). Top enriched genes and commonly enriched genes under two conditions are shown. (G) Kinases targeted by positively enriched sgRNAs in sorted SUSD2 positve cells at passage 3 under XF-LCDM condition. Top enriched genes are shown.

Article Snippet: Lentivirus of the kinome CRISPR library was produced in HEK293T cells by co-transfection of the sgRNA pooled library together with pMD2.G (addgene#12259) and psPAX2 (addgene#12260) using polyethyleneimine (PEI).

Techniques: CRISPR, Knock-Out, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Control, Staining

Journal: Cells

Article Title: Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9

doi: 10.3390/cells14151165

Figure Lengend Snippet: Overview of CRISPR-Cas9-based strategies for generating fluorescent reporter cell lines via in-frame knock-in. ( A ) Schematic illustration of the general strategy for generating fluorescent knock-in reporter cell lines. Cas9-expressing cells are transduced with a gene-specific sgRNA to introduce a double-stranded break at the target locus. A donor DNA template containing a fluorescent reporter is provided for homology-directed repair (HDR), enabling precise insertion of the reporter sequence into the genomic locus of interest. Created with BioRender.com. ( B ) Schematic representation of the dual-plasmid and single-plasmid systems. In the dual-plasmid system, the sgRNA and the donor DNA template are delivered by two separate vectors. The sgRNA plasmid features a doxycycline-inducible sgRNA expression cassette and a miRFP670 fluorescent reporter as a marker for transient expression of the plasmid. The donor DNA vector includes a 5′ homology arm, a fluorescent reporter in frame with a T2A peptide and lacking a start codon, and a 3′ homology arm. In the single-plasmid system, a multiple cloning site (MCS) was engineered into the sgRNA plasmid to enable subcloning of the donor DNA template. Other than expressing sgRNA induced by doxycycline, this plasmid also acts as the donor DNA for HDR within a limited time window after infection. Moreover, cells expressing the miRFP670 reporter from randomly integrated donor DNA can be eliminated by FACS sorting from a few days post-infection onwards. Created with BioRender.com.

Article Snippet: The Human Two Plasmid Activity-Optimized CRISPR Knockout sgRNA Library, developed by David Sabatini, Eric Lander and colleagues, was obtained from Addgene (Cat# 1000000095, Watertown, MA, USA) [ ].

Techniques: CRISPR, Knock-In, Expressing, Transduction, Introduce, Sequencing, Plasmid Preparation, Marker, Cloning, Subcloning, Infection

Journal: Cells

Article Title: Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9

doi: 10.3390/cells14151165

Figure Lengend Snippet: Generation of biallelic knockout cells with fluorescent knock-in reporter as a surrogate of gene disruption. ( A ) Workflow for generating TSPAN8 knock-out cell lines with a GFP knock-in reporter. The donor DNA template consists of an 800 bp 5′ homology arm, T2A-GFP sequence, SV40 polyA signal, and an 800 bp 3′ homology arm flanking exon 2 of the TSPAN8 gene. The TSPAN8 sgRNA target site was abolished in this donor template to prevent further DNA breaks after integration. Cas9-expressing cells were transfected with dual plasmids (sgRNA and donor template) by electroporation. Immediately following transfection, cells were treated with doxycycline (Dox) to induce sgRNA expression for 48 h. After 7–10 days of culture, cells were enriched based on GFP expression levels to isolate targeted populations. ( B ) Representative FACS plot showing the initial sorting of cells. Only cells transfected with TSPAN8 sgRNA exhibit a GFP + population, confirming successful targeting and reporter integration. The GFP + population was gated and sorted for subsequent analyses and enrichment. Both MEC and JHH5 cells were used in this experiment, with 20 µg of total plasmids (1:1 ratio of sgRNA and donor template plasmids) transfected per million cells. ( C ) FACS plot showing TSPAN8 protein expression in relation to GFP expression in the initially sorted cell population (GFP + cells gated in ( B )). Notably, the majority of GFP high cells exhibited an absence of TSPAN8 expression, confirming the efficacy of the reporter enrichment strategy. GFP high cells were sorted to further enrich the homozygous reporter cell population and establish the final cell line. ( D ) FACS analysis demonstrates that most cells in the final homozygous cell line derived from the sorted GFP high population were TSPAN8-negative. ( E ) Western blotting validates the absence of TSPAN8 protein in the homozygous reporter cell line. An established TSPAN8 knockout (KO) and non-targeting (NT) cells generated in our previous study were used as negative and positive controls, respectively, for TSPAN8 expression.

Article Snippet: The Human Two Plasmid Activity-Optimized CRISPR Knockout sgRNA Library, developed by David Sabatini, Eric Lander and colleagues, was obtained from Addgene (Cat# 1000000095, Watertown, MA, USA) [ ].

Techniques: Knock-Out, Knock-In, Disruption, Sequencing, Expressing, Transfection, Electroporation, Derivative Assay, Western Blot, Generated

Journal: Cells

Article Title: Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9

doi: 10.3390/cells14151165

Figure Lengend Snippet: Establishment of biallelic knock-in reporter cells retaining endogenous protein expression. ( A ) Schematic illustration of the targeting strategy for the TSPAN8 gene locus by CRISPR-Cas9. In this strategy, the stop codon of the human TSPAN8 gene is replaced with a T2A-eGFP sequence. The resulting mRNA from the modified allele encodes two separate proteins: TSPAN8 and GFP. ( B ) Representative FACS analysis of electroporated cells. Different dosages of plasmids were tested on MEC cells. Notably, in the absence of TSPAN8 sgRNA (i.e., non-target sgRNA), no GFP-positive cells were observed. ( C ) Representative FACS plots of the reporter cell line generated after multiple rounds of sorting of the GFP high population. The established reporter cell line exhibits high GFP and intact TSPAN8 expression. ( D ) PCR genotyping analysis of the established reporter cell line. The total GFP + cells exhibited two bands corresponding to knock-in (KI) and wild-type (WT) alleles, respectively. The WT allele is absent in the reporter cell line established from the GFP high population.

Article Snippet: The Human Two Plasmid Activity-Optimized CRISPR Knockout sgRNA Library, developed by David Sabatini, Eric Lander and colleagues, was obtained from Addgene (Cat# 1000000095, Watertown, MA, USA) [ ].

Techniques: Knock-In, Expressing, CRISPR, Sequencing, Modification, Generated

Journal: Cells

Article Title: Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9

doi: 10.3390/cells14151165

Figure Lengend Snippet: Generation of knock-in reporter cells using a single-plasmid system integrating both sgRNA and donor DNA. ( A ) Workflow for generating GFP knock-in cell lines using a single plasmid. Cas9-expressing MEC cells were transfected with the single plasmid by electroporation and cultured for 7–10 days. GFP + /miRFP670 − populations were enriched by FACS. ( B ) Representative FACS plots for cells electroporated with different amounts of single plasmid and analysed 10 days post-transduction. Transduced cells with the single plasmid lacking TSPAN8 sgRNA (i.e., non-target sgRNA) were also tested as a negative control. ( C ) Representative FACS plots of the knock-in reporter cell line generated after multiple rounds of GFP high sorting. ( D ) PCR genotyping of the established reporter cell line. The total GFP + cells exhibited bands corresponding to both knock-in (KI) and wild-type (WT) alleles. The WT allele is absent in the reporter cell line derived from the GFP high population.

Article Snippet: The Human Two Plasmid Activity-Optimized CRISPR Knockout sgRNA Library, developed by David Sabatini, Eric Lander and colleagues, was obtained from Addgene (Cat# 1000000095, Watertown, MA, USA) [ ].

Techniques: Knock-In, Plasmid Preparation, Expressing, Transfection, Electroporation, Cell Culture, Transduction, Negative Control, Generated, Derivative Assay

Journal: Cells

Article Title: Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9

doi: 10.3390/cells14151165

Figure Lengend Snippet: Generation of knock-in reporter cells by the IDLV system. ( A ) Workflow for generating GFP knock-in cell lines using the IDLV system. Cas9-expressing MEC cells were infected with lentivirus packaged by the IDLV system. Following infection, cells were treated immediately with doxycycline to induce sgRNA expression. GFP + /miRFP670 − populations were enriched by FACS. ( B ) Representative FACS plots of cells at 10 days post-infection with the indicated amount of lentivirus generated by the IDLV system. Lentivirus lacking sgRNA was used as a negative control. ( C ) Representative FACS plots showing a uniform GFP expression in the established knock-in reporter cells, established by multiple rounds of sorting of the GFP high population. ( D ) Validation of correct integration of GFP reporter into the TSPAN8 locus. Two distinct sgRNAs targeting early exons of TSPAN8 were used for validation. ( E ) PCR analysis of random integration. PCR analysis was conducted using indicated primers targeting the sequences spanning the plasmid backbone and homology arms. Unsorted cells after transduction or the established reporter cell lines by single-plasmid electroporation or IDLV were analysed. ( F ) Droplet digital PCR (ddPCR) analysis of GFP and TSPAN8 was performed to determine their genomic copy numbers. Primers targeting the GFP insert and the endogenous TSPAN8 locus were used to assess the relative abundance of each sequence. Each sample was analysed in triplicate.

Article Snippet: The Human Two Plasmid Activity-Optimized CRISPR Knockout sgRNA Library, developed by David Sabatini, Eric Lander and colleagues, was obtained from Addgene (Cat# 1000000095, Watertown, MA, USA) [ ].

Techniques: Knock-In, Expressing, Infection, Generated, Negative Control, Biomarker Discovery, Plasmid Preparation, Transduction, Electroporation, Digital PCR, Sequencing

Journal: Cells

Article Title: Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9

doi: 10.3390/cells14151165

Figure Lengend Snippet: Genome-wide CRISPR-Cas9 screen for transcriptional regulators of TSPAN8. ( A ) Schematic illustrating the workflow of the genome-scale CRISPR/Cas9 loss-of-function screen to enrich the GFP low cells. JHH5 reporter cells stably expressing Cas9 were transduced with a lentiviral sgRNA library. The GFP low cell population was enriched through repeated rounds of sorting and culture. The enriched sgRNAs were identified via next-generation sequencing (NGS). ( B ) FACS plots showing sequential enrichment of the cells with reduced GFP expression. The GFP low cells were sorted over multiple rounds, with one week of expansion between rounds. ( C ) Candidate genes were identified by comparing the sgRNA read counts between the initial pool and the final GFP low population. ( D ) Scatterplot displaying the sgRNA enrichment for selected candidate genes. ( E – G ) Representative FACS plots showing the reduction in both cell-surface TSPAN8 ( E ) and GFP reporter expression ( G ) following knockout of NF2, DYRK1A, and SOX9. ( H , I ) FACS and qPCR analysis showing that knockout of SOX9 in JHH5 parental cells significantly downregulates TSPAN8 expression. ( J ) Representative FACS plots showing knockout of NF2, DYRK1A, and SOX9 reduced cell-surface TSPAN8 expression in the human HCC cell line SNU878.

Article Snippet: The Human Two Plasmid Activity-Optimized CRISPR Knockout sgRNA Library, developed by David Sabatini, Eric Lander and colleagues, was obtained from Addgene (Cat# 1000000095, Watertown, MA, USA) [ ].

Techniques: Genome Wide, CRISPR, Stable Transfection, Expressing, Transduction, Next-Generation Sequencing, Knock-Out

Journal: Scientific Reports

Article Title: Genome-wide CRISPR screen for human factors involved in alternative polyadenylation based on differential localization of CD47

doi: 10.1038/s41598-025-14782-7

Figure Lengend Snippet: Genome-wide CRISPR screening identifies candidate APA regulators . ( a ) Schematic of the genome-wide CRISPR screen based on differential CD47 localization. ( b ) FACS analysis of cell surface and intracellular CD47 expression in HeLa cells stably expressing Cas9. The 10% of cells with the lowest (CD47 surface high) and 10% with the highest (CD47 surface low) intracellular-to-cell surface CD47 expression ratios were collected. ( c ), ( d ) Gene-level enrichment of sgRNAs in CD47 surface high ( c ) and CD47 surface low ( d ) cells, with the x-axis representing log 2 fold changes and the y-axis showing MAGeCK P-values. Dashed lines indicate P < 0.05 and log 2 fold changes > 1, with significantly enriched genes highlighted. ( e ) ( f ) GO enrichment analysis of top 100 ranked genes whose depletion resulted in cell surface CD47 retention (CD47 surface high) ( e ) or intracellular CD47 accumulation (CD47 surface low) ( f ).

Article Snippet: For pooled genome-wide CRISPR screening, the Guide-it CRISPR Genome-Wide sgRNA Library (Takara), comprising 76,612 sgRNAs targeting 19,114 human genes, was packaged into lentivirus following the manufacturer’s protocol.

Techniques: Genome Wide, CRISPR, Expressing, Stable Transfection

Journal: Scientific Reports

Article Title: Genome-wide CRISPR screen for human factors involved in alternative polyadenylation based on differential localization of CD47

doi: 10.1038/s41598-025-14782-7

Figure Lengend Snippet: Secondary screen identifies candidate genes potentially involved in APA regulation. ( a ) Schematic of the secondary CRISPR screen based on differential CD47 localization. ( b ) Quantification of MFI values from FACS analysis of CD47. The double staining approach was applied to HeLa cells stably expressing control shRNA or shRNA targeting candidate genes. Results are presented as mean ± s.d. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01 (two-tailed t-test for independent samples, n = 3). ( c ) qRT-PCR analysis of total CD47 mRNA and the long 3′ UTR isoform in HeLa cells stably expressing control shRNA or shRNA targeting candidate genes. Results are presented as mean ± s.d. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01 (two-tailed t-test for independent samples, n = 3).

Article Snippet: For pooled genome-wide CRISPR screening, the Guide-it CRISPR Genome-Wide sgRNA Library (Takara), comprising 76,612 sgRNAs targeting 19,114 human genes, was packaged into lentivirus following the manufacturer’s protocol.

Techniques: CRISPR, Double Staining, Stable Transfection, Expressing, Control, shRNA, Two Tailed Test, Quantitative RT-PCR

Journal: Nature Immunology

Article Title: Mechanism for controlled assembly of transcriptional condensates by Aire

doi: 10.1038/s41590-024-01922-w

Figure Lengend Snippet: a , Gating strategy of flow cytometric analyses shown in Fig. . Sample with GFP-P2A-Gal4DBD-CTT expression (+ CTT-gRNA) was shown as an example. b , Sorting strategy of CRISPR screening shown in Fig. . c , LC-MS/MS analysis results of the GST-CTT pull-down in Fig. . A ~ 250 kDa band in the GST-CTT-bound fraction and the equivalent region in the GST control were analyzed. See also Supplementary Table . d , Domain architecture of p300 and the truncation variants used in e . Note that the paralog CBP has the same domain architecture. e , His 6 -GST-CTT variant pull-downs of HA-tagged p300 transiently expressed in 293 T nuclear extracts. Far left: SDS-PAGE gel of GST-CTT variant proteins captured onto glutathione beads used for the pull-down assay. Right: WBs of HA-p300 variants pulled-down with GST-CTT WT vs. GST-CTTΔR2 or GST-CTTΔR3. Data shown are of at least two independent experiments. f , His 6 -GST-CTT variant pull-downs of recombinantly expressed and purified CBP TAZ2. Data is representative of three independent experiments.

Article Snippet: The engineered 4D6 cells were transduced with a lentiviral Human Brunello CRISPR knockout pooled sgRNA library (a gift from David Root and John Doench (Addgene no. 73178-LV) ) at a multiplicity of infection of 0.4, aiming for 500-fold representation of each sgRNA.

Techniques: Expressing, CRISPR, Liquid Chromatography with Mass Spectroscopy, Control, Variant Assay, SDS Page, Pull Down Assay, Purification

Journal: Nature Immunology

Article Title: Mechanism for controlled assembly of transcriptional condensates by Aire

doi: 10.1038/s41590-024-01922-w

Figure Lengend Snippet: a , Top: schematic of Gal4-reporter-based CRISPR screening system in 4D6 cells. Bottom: system validation by flow cytometry. b , sgRNA enrichment in 4D6 cells with decreased (red) versus increased (blue) mKate2 expression (Supplementary Table and Extended Data Fig. ). c , Purified His 6 -GST and His 6 -GST-CTT pull-downs of 293T nuclear extracts. Red arrow, location of ~250-kDa SDS–PAGE gel band cut and analyzed by MS (Extended Data Fig. and Supplementary Table ). d , His 6 -GST and His 6 -GST-CTT pull-downs of HA-p300 from 293T nuclear extracts. e , NMR results incorporated into the top AlphaFold-Multimer model using mouse CBP TAZ2 (aa 1764–1855) and mouse Aire CTT (aa 480–552). CTT is colored by NMR chemical shift perturbation with TAZ2. TAZ2 is colored by electrostatic potential (PyMol, APBS). f , ITC thermograms of CTT titrated into TAZ2 fit to a two-sites binding model. g , His 6 -GST-CTT variant pull-downs of purified FLAG-p300. h , Transcriptional activity of mouse Aire variants in transfected 4D6 cells measured by RT–qPCR (mean ± s.d., three biological replicates). P values (one-way ANOVA with Dunnett’s multiple comparisons test) were calculated versus WT Aire. **** P < 0.0001; NS P > 0.05. i , Representative IF images of 4D6 cells expressing mouse Aire-FLAG variants. Bottom left: percentage of nuclei with Aire condensates versus diffuse Aire ( n , nuclei). Bottom middle: mean fluorescence of Aire-expressing nuclei. Red-boxed nuclei (≤550 a.u.) were selected to compare condensate sizes with similar Aire expression. Bottom right: Aire condensates volume quantification in nuclei with similar mean fluorescence ( n , condensates). P values (Kruskal–Wallis and Dunn’s multiple comparisons test (bottom middle); two-tailed Mann–Whitney test (bottom right)) were calculated versus WT Aire. *** P < 0.001; **** P < 0.0001; NS P > 0.05 (Extended Data Fig. ). j , Aire-induced transcriptional changes in the presence of A-485 versus dimethylsulfoxide (DMSO) within ±20 kb of Aire-bound versus Aire-free NFRs measured by 5EU-seq. k , Aire condensate volume quantification with DMSO, dCBP-1 or A-485 ( n , condensates). P values (Kruskal–Wallis test with Dunn’s multiple comparisons test), **** P < 0.0001; NS P > 0.05 (Extended Data Fig. ). Horizontal lines in i and k indicate the median. a , d , g – i , k are representative of at least three independent experiments. FDR, false discovery rate.

Article Snippet: The engineered 4D6 cells were transduced with a lentiviral Human Brunello CRISPR knockout pooled sgRNA library (a gift from David Root and John Doench (Addgene no. 73178-LV) ) at a multiplicity of infection of 0.4, aiming for 500-fold representation of each sgRNA.

Techniques: CRISPR, Biomarker Discovery, Flow Cytometry, Expressing, Purification, SDS Page, Binding Assay, Variant Assay, Activity Assay, Transfection, Quantitative RT-PCR, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Advances in Virus Research

Article Title: Functional Genomic Strategies for Elucidating Human–Virus Interactions

doi: 10.1016/bs.aivir.2015.11.001

Figure Lengend Snippet: Functional Genomic Screens for Elucidating Host–Viral Interactions

Article Snippet: Next, we stably transduced the H1-HeLa-Cas9 cells at a moi of 0.2 with a complex lentiviral pool expressing the human GeCKO v.2 sgRNA library (Addgene #1000000049), which targets 19,052 genes in the human genome with six sgRNAs per gene across two half-libraries (library A and B) ( ).

Techniques: Functional Assay, Virus, Knockdown, Selection, Biomarker Discovery, Suspension, Mutagenesis, Immunofluorescence, Microscopy, TALENs, Flow Cytometry, Comparison, CRISPR, Recombinant, Expressing, Luciferase, Disruption, Infection, Activity Assay, Inhibition, Western Blot, Genome Wide, In Vivo, Construct, Over Expression, Standard Deviation, Membrane, Staining, Viability Assay, In-Cell ELISA, Transfection, Immunoprecipitation, Control, Northern Blot, Fluorescence, Gene Expression, Microarray, Dominant Negative Mutation, Transmission Assay, Electron Microscopy, Transduction, Amplification, Clone Assay, Enzyme-linked Immunosorbent Assay, Modification, Two Hybrid Assay, Plaque Assay, Single Vesicle Fusion Assay, shRNA, Binding Assay, Sequencing, esiRNA, Glycoproteomics, Blocking Assay, Cell Cycle Assay, Ubiquitin Proteomics, Conjugation Assay, Plasmid Preparation, Mass Spectrometry, Reverse Transcription, Polymerase Chain Reaction

Journal: Advances in Virus Research

Article Title: Functional Genomic Strategies for Elucidating Human–Virus Interactions

doi: 10.1016/bs.aivir.2015.11.001

Figure Lengend Snippet: CRISPR/Cas9 screen for HRV host factors. (A) The HRV-HF CRISPR/Cas9 screen workflow showing the generation of the Cas9 expressing H1-HeLa cells containing the sgRNA libraries followed by their subsequent challenge with HRV14 and the assessment of the enriched sgRNAs using next-gen sequencing. (B) HeLa-H1-Cas9 cells were transduced with Moloney Leukemia virus (MLV)-GFP, then supra-transduced with either an empty vector control (parent population) or one expressing a sgRNA against GFP. The cells were selected for puromycin resistance and cultured for 11 days then fixed and imaged for GFP expression. Differential interference contrast (DIC) images are provided below. 4 × magnification. (C) DIC images of cells transduced with either library A or B that survived the HRV14 challenge were expanded and tested for their susceptibility to HRV14’s cytopathic effect over 2 days (bottom row) compared to the unselected parent cell population and the respective uninfected cell populations (top row). (D) Cells from (C) were fixed and immunostained for ICAM1 surface expression by flow cytometry. (E) A chart showing the relative proportion of total sequencing reads for the recovered sgRNAs from the HRV14 CRISPR/Cas9 pooled screen based upon the analysis of genomic DNA from the surviving cells from library A or B. Gene names are provided for each sgRNA with the associated numbers designating their unique identifying library number.

Article Snippet: Next, we stably transduced the H1-HeLa-Cas9 cells at a moi of 0.2 with a complex lentiviral pool expressing the human GeCKO v.2 sgRNA library (Addgene #1000000049), which targets 19,052 genes in the human genome with six sgRNAs per gene across two half-libraries (library A and B) ( ).

Techniques: CRISPR, Expressing, Sequencing, Transduction, Virus, Plasmid Preparation, Control, Cell Culture, Flow Cytometry

Journal: Cell metabolism

Article Title: ZBTB1 Regulates Asparagine Synthesis and Leukemia Cell Response to L-Asparaginase.

doi: 10.1016/j.cmet.2020.03.008

Figure Lengend Snippet: Figure 1. A CRISPR-Based Genetic Screen Identifies Transcription Machinery Essential for Proliferation under Serine and Asparagine Depri- vation (A) Schematic demonstrating the metabolic genes regulated by ATF4, the effector of the integrated stress response. (B) Schematic depicting pooled CRISPR screens under asparagine or serine deprivation using a transcription-focused sgRNA library. (C) Gene scores in Jurkat cells grown in complete versus serine-deficient media (left). The gene score is the median log2 fold change in the abundance of all sgRNAs targeting a given gene after 14 population doublings. Most genes, as well as non-targeting control sgRNAs, have similar scores in the presence or absence of serine. The top ten genes differentially required under serine deprivation are shown (right). (D) Gene scores in WT Jurkat cells grown in complete versus asparagine-deficient media (left). The top ten genes differentially required under asparagine deprivation are shown (right). Media was depleted of asparagine through the use of L-asparaginase (0.25 U/mL). (E) Changes in the abundance of individual ZBTB1 sgRNAs in complete media (black) or in the absence (gray) of asparagine (top) or serine (bottom). (F) Differential scores of genes required specifically under asparagine deprivation relative to serine deprivation. ZBTB1 is highlighted in blue. (G) Immunoblot analysis of parental, ZBTB1 knockout, and rescued ZBTB1 knockout Jurkat cells (top). b-actin was used as a loading control. (H) Fold change in cell number (log2) of parental (black), ZBTB1 knockout (blue), and rescued ZBTB1 knockout (gray) Jurkat cells after growth in media with indicated asparagine concentrations for 5 days (mean ± SD, n = 3). (I) Fold change in cell number (log2) of parental (black), ZBTB1 knockout (blue), and rescued ZBTB1 knockout (gray) Jurkat cells after treatment with indicated asparaginase concentrations for 5 days (mean ± SD, n = 3) (top). Representative bright-field micrographs of indicated cells after a 5-day asparaginase treatment are shown (bottom). Statistics: two-tailed unpaired t test. **p < 0.05, ***p < 0.01, ****p < 0.001.

Article Snippet: Experimental Models: Organisms/Strains Mouse: NOD scid gamma: IL2Rgammanull The Jackson Laboratory 005557 Oligonucleotides qPCR primers, gBlocks, sgRNA and EMSA oligonucleotides This study See Table S5 Recombinant DNA CRISPR Cas9 sgRNA library This study N/A pLenti CRISPR V2 Addgene 52961 (Continued on next page) Cell Metabolism 31, 852–861.e1–e6, April 7, 2020 e2

Techniques: CRISPR, Control, Western Blot, Knock-Out, Two Tailed Test