Journal: Scientific Reports

Article Title: Sitafloxacin reduces tumor necrosis factor alpha (TNFα) converting enzyme (TACE) phosphorylation and activity to inhibit TNFα release from lipopolysaccharide-stimulated THP-1 cells

doi: 10.1038/s41598-021-03511-5

Figure Lengend Snippet: Sitafloxacin significantly reduced TNFα production. THP-1 cells (2 × 10 5 /mL) were stimulated by LPS (0.1 µg/mL) with several different quinolone antibiotics (50 µg/mL) for 4 h. Data are presented as mean ± SD of 6 independent experiments. *p < 0.05 vs. LPS alone. **p < 0.01 vs. LPS alone. ***p < 0.01 vs. MFLX, LVFX, GRNX, or CPFX. LPS lipopolysaccharide, MFLX moxifloxacin, LVFX levofloxacin, GRNX garenoxacin, CPFX ciprofloxacin, STFX sitafloxacin.

Article Snippet: MFLX, GRNX and CPFX were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Techniques:

Journal: Nanomaterials

Article Title: PLATOX: Integrated In Vitro/In Vivo Approach for Screening of Adverse Lung Effects of Graphene-Related 2D Nanomaterials

doi: 10.3390/nano12081254

Figure Lengend Snippet: GRNP dispersions. Left: Light microscopic pictures of the different GRNP (P1–P8) dispersions at 6.25 µg/mL corresponding to the lowest concentration used for in vitro screening (3.125 µg/cm 2 ). Right: Sedimentation kinetics for the various GRNP (P1–P8) at 6.25 µg/mL. GRNP dispersions were placed in semi-micro cuvettes and turbidity (OD600) was measured directly after material suspension (time point zero) and after 1, 18, 24 and 48 h of incubation at 37 °C in a water saturation atmosphere using an incubator. OD600 at time point zero was set to 100% and relative turbidity was calculated for the other time points. Data represent the means ± SD of three independent experiments.

Article Snippet: Six GRNP samples, including three functionalized materials, were purchased from ACS Material, LLC (Medford, MA, USA).

Techniques: Concentration Assay, In Vitro, Sedimentation, Suspension, Incubation

Journal: Nanomaterials

Article Title: PLATOX: Integrated In Vitro/In Vivo Approach for Screening of Adverse Lung Effects of Graphene-Related 2D Nanomaterials

doi: 10.3390/nano12081254

Figure Lengend Snippet: GRNP-related histopathological findings at day 1 post-exposure.

Article Snippet: Six GRNP samples, including three functionalized materials, were purchased from ACS Material, LLC (Medford, MA, USA).

Techniques:

Journal: Nanomaterials

Article Title: PLATOX: Integrated In Vitro/In Vivo Approach for Screening of Adverse Lung Effects of Graphene-Related 2D Nanomaterials

doi: 10.3390/nano12081254

Figure Lengend Snippet: GRNP-related histopathological findings at day 29 post-exposure.

Article Snippet: Six GRNP samples, including three functionalized materials, were purchased from ACS Material, LLC (Medford, MA, USA).

Techniques:

Journal: Science signaling

Article Title: Phagocytosed photoreceptor outer segments activate mTORC1 in the retinal pigment epithelium

doi: 10.1126/scisignal.aag3315

Figure Lengend Snippet: (A) POS-induced mTORC1 activation in ARPE-19 cells transfected with either KLC1 small interfering RNA (siRNA) or control scrambled (Scr) siRNA. S6 phosphorylation and KLC1 knockdown were analyzed by Western blots. Quantification data are means from five independent experiments (mean ± SEM). *P<0.05 (Kruskal-Wallis test and Dunn’s multiple comparisons test). (B) and (C) Co- immunostaining of rhodopsin and myosin 6 (Myo6) in POS-treated ARPE-19 cells and RPE/choroid whole mounts, respectively. Images are representative of three independent experiments. Scale bar: 5 μm. (D) Quantitation of the data in B and C. Twenty fields with an area of 50 ϗ 50 μm2 were analyzed. (E) Depletion of Myo6 in ARPE-19 cells expressing Cas9 endonuclease and transfected with gRNA targeting Myo6. Blots are representative of three independent experiments. (F) Degradation of POS in cells depleted of Myo6 by CRISPR/Cas9. Quantification data are means from six independent experiments (mean ± SEM). *P<0.05 (Kruskal-Wallis test and Dunn’s multiple comparisons test). (G) POS-induced mTORC1 activation in cells depleted of Myo6 by CRISPR/Cas9. Quantification data are means from five independent experiments (mean ± SEM). ***P<0.001 (one-way ANOVA and Tukey- Kramer Multiple comparsions Test). (H) Amino Acid (AA)-induced mTORC1 activation in cells depleted of Myo6 by CRISPR/Cas9. Data are means of five independent experiments (mean ± SEM). ***P<0.001 (one-way ANOVA and Tukey-Kramer Multiple comparsions Test).

Article Snippet: Lentivirus-expressing guide RNA (gRNA) targeting Myo6 9 (pLV[gRNA]-Puro-U6>hMY06) or scrambled gRNA (pLV[gRNA]-Puro-U6>Scramblc_gRNA) were designed, cloned, and packaged by VectorBuilder.

Techniques: Activation Assay, Transfection, Small Interfering RNA, Control, Phospho-proteomics, Knockdown, Western Blot, Immunostaining, Quantitation Assay, Expressing, CRISPR

Journal: Blood Advances

Article Title: Large-scale GMP-compliant CRISPR-Cas9–mediated deletion of the glucocorticoid receptor in multivirus-specific T cells

doi: 10.1182/bloodadvances.2020001977

Figure Lengend Snippet: CRISPR-Cas9–mediated deletion of the gene coding for the GR in primary human VSTs. (A) Schematic summary of the protocol for CRISPR-Cas9–mediated KO of NR3C1 in primary human VSTs. PBMCs were stimulated with virus-specific PepMix from CMV, BKV, and adenovirus (in combination) in the presence of 10 ng/mL IL-7, 50 IU/mL IL-2, and 10 ng/mL IL-15. On days 7 to 10 of in vitro expansion, primary human VSTs were nucleofected with control Cas9 alone (Cas9 control) or Cas9 preloaded with gRNA targeting the exon 2 of the NR3C1 gene, which encodes for the GR protein. KO efficiency and functional assays were performed at day +14 post initial PBMC isolation. (B) Schematic representation of CRISPR-Cas9–mediated NR3C1 KO using 2 short guide crRNAs targeting the exon 2 of NR3C1 gene. (C-D) The NR3C1 KO efficiency of VSTs after electroporation with Cas9 alone (control), Cas9 complexed with 1 crRNA (crRNA 1 or crRNA 2), or Cas9 complexed with the combination of 2 crRNAs (crRNA 1 plus crRNA 2) using a WT Cas9 was determined by PCR analysis at day 3 (C) or western blot analysis at day 7 (D) after electroporation. The percentages (%) of NR3C1 KO efficiency (C) and GR protein loss (D) after CRISPR-Cas9 gene editing are shown under each figure. PAM, protospacer adjacent motif.

Article Snippet: On days 7 to 10 of expansion, different VST numbers (3 × 10 6 , 25 × 10 6 , and 100 × 10 6 ) were electroporated in the presence of Cas9 and gRNA using the Lonza 4D nucleofector.

Techniques: CRISPR, In Vitro, Functional Assay, Isolation, Electroporation, Western Blot

Journal: Blood Advances

Article Title: Large-scale GMP-compliant CRISPR-Cas9–mediated deletion of the glucocorticoid receptor in multivirus-specific T cells

doi: 10.1182/bloodadvances.2020001977

Figure Lengend Snippet: Identification of Cas9 off-target sites by GUIDE-seq and quantification of potential Cas9 off-target cleavage sites using rhAmpSeq technology. (A) Sequences of off-target sites identified by GUIDE-seq for 2 gRNAs targeting the NR3C1 locus (gRNA#1, top panel; gRNA#2, bottom panel). The guide sequence is listed on top with off-target sites shown below. The on-target site is identified with a black square. Mismatches to the guide are shown and highlighted in color with insertions shown in gray below. The number of GUIDE-seq sequencing reads are shown to the right of each site. Pie charts indicate the fractional percentage of the total unique, CRISPR-Cas9–specific read counts that are on-target (orange) and off-target (blue). (B) On- and off-target effects were determined by targeted amplification followed by NGS for exon 2–guide 1 and for exon 2–guide 2. Individual gRNAs were delivered into HEK293 cells stably expressing WT-Cas9 (left panels), or delivered into HEK293 cells by complexing to WT-Cas9 protein (middle panels) or HiFi-Cas9 protein (right panels). Pie charts indicate the percentage of on-target effect (in red) and off-target effect (in blue). (C) Editing efficiency was determined using targeted amplification followed by NGS. Exon 2–guide 1 and exon 2–guide 2 gRNAs were complexed simultaneously with either WT-Cas9 (blue bars) or HiFi-Cas9 (red bars) into HEK293 cells. Editing efficiencies were determined for the known on- and off-target sites for exon 2–guide 1 (left top panel), and exon 2–guide 2 (right top panel). In a similar experiment, exon 2–guide 1 and exon 2–guide 2 gRNAs were complexed simultaneously with either WT-Cas9 (blue bars) or HiFi-Cas9 (red bars) into primary human T cells. Editing efficiencies were determined for the known on- and off-target sites for exon 2–guide 1 (left bottom panel), and exon 2–guide 2 (right bottom panel). Data are shown as mean and SD from n = 3 human donors.

Article Snippet: On days 7 to 10 of expansion, different VST numbers (3 × 10 6 , 25 × 10 6 , and 100 × 10 6 ) were electroporated in the presence of Cas9 and gRNA using the Lonza 4D nucleofector.

Techniques: Sequencing, CRISPR, Amplification, Stable Transfection, Expressing

Journal: Blood Advances

Article Title: Large-scale GMP-compliant CRISPR-Cas9–mediated deletion of the glucocorticoid receptor in multivirus-specific T cells

doi: 10.1182/bloodadvances.2020001977

Figure Lengend Snippet: Successful scale-up of CRISPR-Cas9–mediated NR3C1 deletion in VSTs. (A) Different VST numbers (3 × 106, 25 × 106, and 100 × 106) were electroporated in the presence of Cas9 and gRNA using the Lonza 4D nucleofector and the NR3C1 KO efficiency in Cas9 (control) or NR3C1 KO VSTs was determined using PCR (A; n = 3) and western blot analysis (B; n = 3). β-actin was used as loading control in panel B. The percentages (%) of NR3C1 KO efficiency (A) and GR protein loss (B) after CRISPR-Cas9 gene editing are shown under each figure. (C) Representative FACS plots showing the percentage of apoptotic cells (annexin V+) and alive or dead cells (live/dead stain) in control Cas9 and NR3C1 KO VST cells at the different cell dose levels of 3 × 106, 25 × 106, or 100 × 106 cells per electroporation treated with or without Dexa (200 μM) for 72 hours (n = 3). (D) Graph summarizing the absolute cell number between control Cas9 (solid lines) and NR3C1 KO VST cells (dotted lines) at the different cell dose levels of 3 × 106 (green), 25 × 106 (blue), or 100 × 106 (red) cells per electroporation for 72 hours (n = 3). Bars represent mean values with SD. (E-F) Bar graphs showing the percentage of IFN-γ, TNF-α, or IL-2 production by 100 × 106 control Cas9 (green), 100 × 106 NR3C1 KO (blue), or 100 × 106 NR3C1 KO plus dexamethasone (Dexa 200 μM; red) VSTs in response to 6-hour stimulation with the relevant viral PepMix in the CD8+ T-cell (E) and CD4+ T-cell (F) compartments (n = 3). The functional analysis of the Cas9+Dexa group was not performed due to the absence of viable cells resulting from the lymphocytotoxic effect of steroids. The bars represent mean values with SD.

Article Snippet: On days 7 to 10 of expansion, different VST numbers (3 × 10 6 , 25 × 10 6 , and 100 × 10 6 ) were electroporated in the presence of Cas9 and gRNA using the Lonza 4D nucleofector.

Techniques: CRISPR, Western Blot, Staining, Electroporation, Functional Assay

Journal: Microbial Cell Factories

Article Title: Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis

doi: 10.1186/s12934-022-01896-0

Figure Lengend Snippet: Plasmid design and construction of the all-in-one plasmid CRISPR-Cas9 system. a Assembly of spacer and donar DNA. The gRNA target under the constitutive promoters for genome editing. The another gRNA rep under the inducible promoter for plasmid curing. b Squence of gRNA rep under the control of P acoR promoter. c Strategy for iterative genome editing in B. subtilis . In the genome editing phase, sgRNA target /Cas9 complex cuts the genome for homologous recombination. In the plasmid curing phase, sgRNA rep /Cas9 complex tragets the repA gene to eliminate the editing plasmid. d Tetracycline sensitivity test for the plasmid self-curing system, 16 clones were picked for tetracycline sensitivity test, and only one single clone showed tetracycline resistance

Article Snippet: P glyA - Bsa I- Nde I- Bsa I-gRNA scaffold-T1 terminator and P acoR -gRNA rep -gRNA scaffold fragments were synthesized by GenScript (Piscataway, NJ).

Techniques: Plasmid Preparation, CRISPR, Control, Homologous Recombination, Clone Assay

Journal: Microbial Cell Factories

Article Title: Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis

doi: 10.1186/s12934-022-01896-0

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: P glyA - Bsa I- Nde I- Bsa I-gRNA scaffold-T1 terminator and P acoR -gRNA rep -gRNA scaffold fragments were synthesized by GenScript (Piscataway, NJ).

Techniques: Preserving, Plasmid Preparation, Expressing

Journal: Nature

Article Title: Systematic discovery of CRISPR-boosted CAR T cell immunotherapies

doi: 10.1038/s41586-025-09507-9

Figure Lengend Snippet: a , Overview of the CELLFIE platform for highly scalable CAR T cell engineering and CRISPR screening. Human primary T cells are isolated, activated and expanded, transduced with a lentivirus carrying sequences for the CAR and gRNA library and electroporated with synthetic mRNA delivering the CRISPR editor. The CRISPR-edited CAR T cells are functionally screened in vitro and in vivo using multiple readouts. hU6, human U6 promoter; LTR, long terminal repeat; puro, puromycin. b , Experimental timeline for genome-wide fitness screens. Human primary T cells are isolated from whole blood, activated, pre-expanded, activated again and transduced with the CROP-seq-CAR lentivirus for co-delivery of sequences for the CAR and the genome-wide gRNA library. Two days later, cells are electroporated with synthetic mRNA for Cas9 and blasticidin resistance (blasticidin-S deaminase, BSD), followed by antibiotic selection for successful lentiviral transduction (puromycin) and successful mRNA electroporation (blasticidin). CRISPR-edited CAR T cells are expanded under repeated TCR stimulation with anti-CD3/CD28 beads or repeated CAR stimulation with CD19 + K562 cells. Genomic DNA is collected, and gRNA representation is analysed by sequencing on days 0, 7, 14 and 21. c , Gene-level log 2 [fold change (FC)] between day 14 and day 0 for the fitness screens (four donors in two independent screens), mapped onto a schematic of the TCR and CAR signalling pathways. For each protein shape, the colour in the top half corresponds to the TCR stimulation screens and the colour in the bottom half corresponds to the CAR stimulation screens. d , Effect sizes for fitness screens with TCR stimulation ( x axis) and CAR stimulation ( y axis). MAGeCK MLE β values comparing day 14 and day 0 are normalized to values for essential genes to account for the different proliferation rates upon TCR or CAR stimulation. Colours denote screening hits with increased fitness (green), known negative T cell regulators (magenta), known essential genes (purple) and neutral olfactory receptors (blue).

Article Snippet: For RNP electroporation, synthetic gRNA (TrueGuide Synthetic gRNA, Synthego) and Alt-R S.p.

Techniques: CRISPR, Isolation, Transduction, In Vitro, In Vivo, Genome Wide, Selection, Electroporation, Sequencing

Journal: Nature

Article Title: Systematic discovery of CRISPR-boosted CAR T cell immunotherapies

doi: 10.1038/s41586-025-09507-9

Figure Lengend Snippet: a , Titration of synthetic mRNA concentration for efficient CRISPR knockout of CD44 in human primary CD4 + and CD8 + T cells (2 donors), with pan-CD3 + T cells as starting material. Comparable editing efficiencies were observed for custom-made and commercial Cas9 mRNA. b , Consistent editing efficiencies in CD4 + T cells for custom-made Cas9 mRNA from multiple production rounds. c , Cell proliferation of CD4 + and CD8 + CAR T cells made from pan-CD3 + T cells (green), from isolated CD4 + T cells (blue), or from isolated CD8 + T cells (purple) as starting material. d , Declining representation of CD4 + CAR T cells in co-culture with CD8 + CAR T cells when using pan-CD3 + T cells as starting material. e , Puromycin titration to determine an optimal concentration (0.5 µg ml −1 , grey dotted line) to select for T cells that have been successfully transduced with the CROP-seq-CAR lentivirus. f , Blasticidin titration to determine an optimal concentration (50 µg ml −1 , grey dotted line) to select for T cells that were successfully electroporated with blasticidin resistance mRNA co-delivered with the CRISPR editor mRNA. g , CD44 knockout efficiency using the optimized electroporation programs for small-scale experiments (up to 1.5 million cells per cuvette, Amaxa electroporator) and large screens (up to 100 million cells per cuvette, MaxCyte electroporator). Results are shown for CD4 + and CD8 + T cells from pan-CD3 + T cells (1 donor). h , T cell expansion after electroporation of synthetic Cas9 mRNA and blasticidin resistance mRNA (1 donor). i , Effect of T cell stimulation reagents on lentiviral transduction rates (4 donors). j , Effect of common transduction supplements on lentiviral transduction rates (4 donors). k , Effect of common transduction supplements on T cell viability (4 donors). l , Quantification of lentivirus titers using RT-qPCR of lentiviral RNA (mean ± s.e.m. for 3 technical replicates). m , Percent transduced CD4 + and CD8 + T cells for a titration of lentivirus amounts. The chosen amounts (CD4 + : 247 copies per cell; CD8 + : 432 copies per cell) are indicated by dotted lines (2 donors). n , Detection of gRNAs (y-axis: gRNA read counts) in clonally expanded human primary T cells transduced with CROP-seq-CAR lentivirus carrying the genome-wide Brunello gRNA library. Representative examples of T cell clones with 1, 2, or 3 gRNA integrations are shown, and a total of 62 clonally expanded T cell clones were profiled. o , Barplot showing the frequency of 1, 2, or 3 independent lentiviral integrations into the same cell across 62 clonally expanded T cell clones. The average number of gRNA integrations per cell was 1.5. p , CAR expression in human primary CAR T cells prepared with the CROP-seq-CAR lentivirus, using PE-labelled recombinant CD19 antigen for labeling. q , Specific killing of CD19 + cancer cells by CAR T cells prepared with the CROP-seq-CAR (anti-CD19) lentivirus. For all boxplots (panels i-l ), the center line is the median, the box limits are the upper and lower quartiles, and the whiskers extend to 1.5 times the interquartile range.

Article Snippet: For RNP electroporation, synthetic gRNA (TrueGuide Synthetic gRNA, Synthego) and Alt-R S.p.

Techniques: Titration, Concentration Assay, CRISPR, Knock-Out, Isolation, Co-Culture Assay, Transduction, Electroporation, Cell Stimulation, Quantitative RT-PCR, Genome Wide, Clone Assay, Expressing, Recombinant, Labeling

Journal: Nature

Article Title: Systematic discovery of CRISPR-boosted CAR T cell immunotherapies

doi: 10.1038/s41586-025-09507-9

Figure Lengend Snippet: a , Proof-of-concept fitness screen with a focused gRNA library of 100 gRNAs. gRNA-level log fold changes are shown for positive control gRNAs (targeting the puromycin resistance gene PAC) and control gRNAs (targeting a safe harbor locus) at days 7, 14, or 21 relative to day 0. Cells were electroporated with custom-made (top) or commercial (bottom) Cas9 mRNA. b , gRNA representation after cloning the genome-wide Brunello library into the CROP-seq-CAR vector, plotted as a cumulative distribution function based on amplicon sequencing of the plasmid pool, with highlighted fold difference between the 10 th and 90 th percentiles as a measure of gRNA library balance. c , Detailed experimental timeline for the genome-wide fitness screens (4 donors, 2 independent experiments). Key steps in the CELLFIE workflow are highlighted. Samples for gRNA sequencing were collected at day 0 (before Cas9 electroporation), and at days 7, 14, and 21 after electroporation. d , Expansion of human primary CAR T cells upon repeated CAR or TCR stimulation (2 donors). e , CD19 accumulation on the surface of CAR T cells during co-culture with target cells as the result of trogocytosis. f , Flow cytometry profiling of the T cell exhaustion markers PD1, LAG3, TIM3, and TIGIT during co-culture of CAR T cells with K562-CD19 target cells. g , T cell subset profiling by flow cytometry at the isolation and readout time points of the genome-wide screens. h , Essential genes in human primary CAR T cells under CAR and TCR stimulation (4 donors, details in Supplementary Table ). The scatterplot shows gene-level log fold changes comparing day 14 and day 0 of the screen (x-axis) plotted against FDR-adjusted p-values (y-axis). Genes that passed stringent significance thresholds (FDR < 0.01 and log FC < − 1.5 based on MAGeCK RRA) are highlighted. i , Gene set enrichment analysis for essential genes in human primary CAR T cells under CAR and TCR stimulation. Clustering of the top-100 most enriched Gene Ontology (GO) terms from the Biological Process category (left) is shown together with one cluster related to T cell functions visualized as a tree plot (right).

Article Snippet: For RNP electroporation, synthetic gRNA (TrueGuide Synthetic gRNA, Synthego) and Alt-R S.p.

Techniques: Positive Control, Control, Cloning, Genome Wide, Plasmid Preparation, Amplification, Sequencing, Electroporation, Co-Culture Assay, Flow Cytometry, Isolation

Journal: Nature

Article Title: Systematic discovery of CRISPR-boosted CAR T cell immunotherapies

doi: 10.1038/s41586-025-09507-9

Figure Lengend Snippet: a , Experimental timeline of the in vivo validation experiments with CRISPR-boosted CAR T cells, which were genetically engineered either by lentiviral co-delivery of the CAR and a pool of 8 gRNAs followed by mRNA delivery of Cas9 (as in the in vitro screens, top) or by electroporation of a pre-assembled RNP complex of Cas9 protein and one top-performing gRNA (as is common practice in CRISPR-edited cell therapy, bottom). b , CAR T cell titration in a xenograft mouse model of human leukemia. Immunodeficient NSG mice were injected with 0.5 million NALM6 cells engineered to express firefly luciferase. On day 5, mice were treated with different doses of CAR T cells. Leukemic cell load was monitored using live bioluminescence imaging. When left untreated, mice succumb to the leukemia around day 21. To make the model most informative, we selected a low (and deliberately non-curative) dose of CAR T cells that leads to initial leukemic control followed by a quick relapse. c , Survival analysis for the mice shown in panel b . d , Percentage of UMI reads perfectly matching the reference sequence, comparing the established method (gRNA amplification from genomic DNA) with in vivo CROP-seq (gRNA amplification from mRNA), both tested with single PCR and nested PCR amplification. e , Optimal number of UMI-based internal replicates for data analysis based on screening controls. Given that each UMI base can be A, C, G, or T, using UMI bases 1 to 5 results in 4, 16, 64, 256, and 1024 random internal replicates. Standard analysis is labeled as 0 internal replicates (left). Negative, neutral, and positive controls are color-coded. f , Dropout of neutral control gRNAs targeting a safe harbor locus when the read number in the internal replicates gets too small for large numbers of internal replicates. The box plot’s center line indicates the median, the box limits represent the upper and lower quartiles, and the whiskers extend to 1.5 times the interquartile range. g , Recall of negative and positive controls for different numbers of internal replicates. The grey line represents the optimal number of 16 internal replicates chosen for the analysis. h , gRNAs selected for in vivo screens (8 gRNAs per gene) or individual validation (as pools of 8 or single gRNAs). i , Individual effects of the 8 gRNAs per gene in the focused validation screen, with single gRNAs used for individual validation highlighted. The box plot’s center line indicates the median, the box limits are the upper and lower quartiles, and the whiskers extend to 1.5 times the interquartile range. j , Number of distinct T cell clones detected based on unique molecular identifiers (UMIs) in the in vivo screens. k , Estimation of CELLFIE’s scalability to large discovery screens in vivo, extrapolating the number of screenable perturbations from empirical measurements for different screening configurations.

Article Snippet: For RNP electroporation, synthetic gRNA (TrueGuide Synthetic gRNA, Synthego) and Alt-R S.p.

Techniques: In Vivo, Biomarker Discovery, CRISPR, In Vitro, Electroporation, Titration, Injection, Luciferase, Imaging, Control, Sequencing, Amplification, Nested PCR, Labeling, Clone Assay