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a , In vitro transcription to generate TruEditor mRNAs for high efficiency and low immunogenicity delivery. b , Representative flow cytometry plots from HUES66-GFP human embryonic stem cells to measure precise gene editing via GFP-to-BFP conversion. c , Precise gene editing in HUES66-GFP human embryonic stem cells nucleofected with TruEditor <t>mRNA,</t> GFP-targeting gRNA, and a single-stranded repair template encoding BFP-specific edits ( n = 3 biological replicates). d , Representative native fluorescent protein and immunofluorescence images from HUES66 human embryonic stem cells. Scale bar, 50 μm. e , Knock-in of a 2.8 kb anti-CD19 chimeric antigen receptor (CAR) into the TRAC locus in primary CD8+ human T cells and quantification of editing and tumor killing ( n = 2 human donors). f , Representative flow cytometry plots to measure CAR knock in and TCR loss. g , Quantification of CAR knock-in and TCR loss in primary human T cells by flow cytometry ( n = 2 human donors with 3 nucleofection replicates each) h , Representative images from primary CD8+ human T cells edited as in f,g after 96 hours in co-culture with CD19+ Nalm6-GFP leukemia cells (effector:target [E:T] ratio of 1:2). Scale bar, 100 μm. i , Normalized GFP intensity for CD19+ Nalm6-GFP leukemia cells in co-culture with primary CD8+ human T cells edited as in f,g ( n = 24 images per condition with 4 images per nucleofection and 3 nucleofections for each of 2 human donors). For all comparisons, statistical significance was determined using a one-way ANOVA with Tukey’s HSD post hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001).
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a , In vitro transcription to generate TruEditor mRNAs for high efficiency and low immunogenicity delivery. b , Representative flow cytometry plots from HUES66-GFP human embryonic stem cells to measure precise gene editing via GFP-to-BFP conversion. c , Precise gene editing in HUES66-GFP human embryonic stem cells nucleofected with TruEditor <t>mRNA,</t> GFP-targeting gRNA, and a single-stranded repair template encoding BFP-specific edits ( n = 3 biological replicates). d , Representative native fluorescent protein and immunofluorescence images from HUES66 human embryonic stem cells. Scale bar, 50 μm. e , Knock-in of a 2.8 kb anti-CD19 chimeric antigen receptor (CAR) into the TRAC locus in primary CD8+ human T cells and quantification of editing and tumor killing ( n = 2 human donors). f , Representative flow cytometry plots to measure CAR knock in and TCR loss. g , Quantification of CAR knock-in and TCR loss in primary human T cells by flow cytometry ( n = 2 human donors with 3 nucleofection replicates each) h , Representative images from primary CD8+ human T cells edited as in f,g after 96 hours in co-culture with CD19+ Nalm6-GFP leukemia cells (effector:target [E:T] ratio of 1:2). Scale bar, 100 μm. i , Normalized GFP intensity for CD19+ Nalm6-GFP leukemia cells in co-culture with primary CD8+ human T cells edited as in f,g ( n = 24 images per condition with 4 images per nucleofection and 3 nucleofections for each of 2 human donors). For all comparisons, statistical significance was determined using a one-way ANOVA with Tukey’s HSD post hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001).
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a , In vitro transcription to generate TruEditor mRNAs for high efficiency and low immunogenicity delivery. b , Representative flow cytometry plots from HUES66-GFP human embryonic stem cells to measure precise gene editing via GFP-to-BFP conversion. c , Precise gene editing in HUES66-GFP human embryonic stem cells nucleofected with TruEditor <t>mRNA,</t> GFP-targeting gRNA, and a single-stranded repair template encoding BFP-specific edits ( n = 3 biological replicates). d , Representative native fluorescent protein and immunofluorescence images from HUES66 human embryonic stem cells. Scale bar, 50 μm. e , Knock-in of a 2.8 kb anti-CD19 chimeric antigen receptor (CAR) into the TRAC locus in primary CD8+ human T cells and quantification of editing and tumor killing ( n = 2 human donors). f , Representative flow cytometry plots to measure CAR knock in and TCR loss. g , Quantification of CAR knock-in and TCR loss in primary human T cells by flow cytometry ( n = 2 human donors with 3 nucleofection replicates each) h , Representative images from primary CD8+ human T cells edited as in f,g after 96 hours in co-culture with CD19+ Nalm6-GFP leukemia cells (effector:target [E:T] ratio of 1:2). Scale bar, 100 μm. i , Normalized GFP intensity for CD19+ Nalm6-GFP leukemia cells in co-culture with primary CD8+ human T cells edited as in f,g ( n = 24 images per condition with 4 images per nucleofection and 3 nucleofections for each of 2 human donors). For all comparisons, statistical significance was determined using a one-way ANOVA with Tukey’s HSD post hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001).
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a , In vitro transcription to generate TruEditor mRNAs for high efficiency and low immunogenicity delivery. b , Representative flow cytometry plots from HUES66-GFP human embryonic stem cells to measure precise gene editing via GFP-to-BFP conversion. c , Precise gene editing in HUES66-GFP human embryonic stem cells nucleofected with TruEditor <t>mRNA,</t> GFP-targeting gRNA, and a single-stranded repair template encoding BFP-specific edits ( n = 3 biological replicates). d , Representative native fluorescent protein and immunofluorescence images from HUES66 human embryonic stem cells. Scale bar, 50 μm. e , Knock-in of a 2.8 kb anti-CD19 chimeric antigen receptor (CAR) into the TRAC locus in primary CD8+ human T cells and quantification of editing and tumor killing ( n = 2 human donors). f , Representative flow cytometry plots to measure CAR knock in and TCR loss. g , Quantification of CAR knock-in and TCR loss in primary human T cells by flow cytometry ( n = 2 human donors with 3 nucleofection replicates each) h , Representative images from primary CD8+ human T cells edited as in f,g after 96 hours in co-culture with CD19+ Nalm6-GFP leukemia cells (effector:target [E:T] ratio of 1:2). Scale bar, 100 μm. i , Normalized GFP intensity for CD19+ Nalm6-GFP leukemia cells in co-culture with primary CD8+ human T cells edited as in f,g ( n = 24 images per condition with 4 images per nucleofection and 3 nucleofections for each of 2 human donors). For all comparisons, statistical significance was determined using a one-way ANOVA with Tukey’s HSD post hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001).
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a , In vitro transcription to generate TruEditor mRNAs for high efficiency and low immunogenicity delivery. b , Representative flow cytometry plots from HUES66-GFP human embryonic stem cells to measure precise gene editing via GFP-to-BFP conversion. c , Precise gene editing in HUES66-GFP human embryonic stem cells nucleofected with TruEditor <t>mRNA,</t> GFP-targeting gRNA, and a single-stranded repair template encoding BFP-specific edits ( n = 3 biological replicates). d , Representative native fluorescent protein and immunofluorescence images from HUES66 human embryonic stem cells. Scale bar, 50 μm. e , Knock-in of a 2.8 kb anti-CD19 chimeric antigen receptor (CAR) into the TRAC locus in primary CD8+ human T cells and quantification of editing and tumor killing ( n = 2 human donors). f , Representative flow cytometry plots to measure CAR knock in and TCR loss. g , Quantification of CAR knock-in and TCR loss in primary human T cells by flow cytometry ( n = 2 human donors with 3 nucleofection replicates each) h , Representative images from primary CD8+ human T cells edited as in f,g after 96 hours in co-culture with CD19+ Nalm6-GFP leukemia cells (effector:target [E:T] ratio of 1:2). Scale bar, 100 μm. i , Normalized GFP intensity for CD19+ Nalm6-GFP leukemia cells in co-culture with primary CD8+ human T cells edited as in f,g ( n = 24 images per condition with 4 images per nucleofection and 3 nucleofections for each of 2 human donors). For all comparisons, statistical significance was determined using a one-way ANOVA with Tukey’s HSD post hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001).
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a , In vitro transcription to generate TruEditor mRNAs for high efficiency and low immunogenicity delivery. b , Representative flow cytometry plots from HUES66-GFP human embryonic stem cells to measure precise gene editing via GFP-to-BFP conversion. c , Precise gene editing in HUES66-GFP human embryonic stem cells nucleofected with TruEditor mRNA, GFP-targeting gRNA, and a single-stranded repair template encoding BFP-specific edits ( n = 3 biological replicates). d , Representative native fluorescent protein and immunofluorescence images from HUES66 human embryonic stem cells. Scale bar, 50 μm. e , Knock-in of a 2.8 kb anti-CD19 chimeric antigen receptor (CAR) into the TRAC locus in primary CD8+ human T cells and quantification of editing and tumor killing ( n = 2 human donors). f , Representative flow cytometry plots to measure CAR knock in and TCR loss. g , Quantification of CAR knock-in and TCR loss in primary human T cells by flow cytometry ( n = 2 human donors with 3 nucleofection replicates each) h , Representative images from primary CD8+ human T cells edited as in f,g after 96 hours in co-culture with CD19+ Nalm6-GFP leukemia cells (effector:target [E:T] ratio of 1:2). Scale bar, 100 μm. i , Normalized GFP intensity for CD19+ Nalm6-GFP leukemia cells in co-culture with primary CD8+ human T cells edited as in f,g ( n = 24 images per condition with 4 images per nucleofection and 3 nucleofections for each of 2 human donors). For all comparisons, statistical significance was determined using a one-way ANOVA with Tukey’s HSD post hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Journal: bioRxiv

Article Title: Rational design of synthetic proteins using a genome-scale CRISPR screen

doi: 10.64898/2026.02.19.706875

Figure Lengend Snippet: a , In vitro transcription to generate TruEditor mRNAs for high efficiency and low immunogenicity delivery. b , Representative flow cytometry plots from HUES66-GFP human embryonic stem cells to measure precise gene editing via GFP-to-BFP conversion. c , Precise gene editing in HUES66-GFP human embryonic stem cells nucleofected with TruEditor mRNA, GFP-targeting gRNA, and a single-stranded repair template encoding BFP-specific edits ( n = 3 biological replicates). d , Representative native fluorescent protein and immunofluorescence images from HUES66 human embryonic stem cells. Scale bar, 50 μm. e , Knock-in of a 2.8 kb anti-CD19 chimeric antigen receptor (CAR) into the TRAC locus in primary CD8+ human T cells and quantification of editing and tumor killing ( n = 2 human donors). f , Representative flow cytometry plots to measure CAR knock in and TCR loss. g , Quantification of CAR knock-in and TCR loss in primary human T cells by flow cytometry ( n = 2 human donors with 3 nucleofection replicates each) h , Representative images from primary CD8+ human T cells edited as in f,g after 96 hours in co-culture with CD19+ Nalm6-GFP leukemia cells (effector:target [E:T] ratio of 1:2). Scale bar, 100 μm. i , Normalized GFP intensity for CD19+ Nalm6-GFP leukemia cells in co-culture with primary CD8+ human T cells edited as in f,g ( n = 24 images per condition with 4 images per nucleofection and 3 nucleofections for each of 2 human donors). For all comparisons, statistical significance was determined using a one-way ANOVA with Tukey’s HSD post hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Finally, mRNA was purified using a Monarch mRNA purification kit (NEB T2050L).

Techniques: In Vitro, Immunopeptidomics, Flow Cytometry, Immunofluorescence, Knock-In, Co-Culture Assay