Fig 1: Optimization of sgRNA scaffolds for orthogonal CRISPRa/i in hiPSCs(A) Flow cytometry quantification of dCas9–2A-mCherry expression in WTC11 hiPSCs carrying a randomly integrated transgene, hereafter Cas9-R, compared with parental wild-type (WT) hiPSCs.(B) RT-qPCR quantification of dCas9 expression in Cas9-R hiPSCs. N = 3 successive passages; p = 0.0104 versus WT by unpaired two-tailed Welch’s t-test (*).(C) Workflow for sgRNA-scaffold optimization. Cas9-R hiPSCs were targeted at the AAVS1 locus with cassettes encoding various sgRNA scaffolds and matched RBP–effector modules for CRISPRa or CRISPRi testing. HAR, homology arm; PuroR, puromycin N-acetyltransferase; Poly(A), bovine growth hormone polyadenylation signal; U6-TETO2, Pol III U6 promoter with a tet operator; CAG, cytomegalovirus immediate early enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter; RBP, RNA-binding protein; T2A-P2A, tandem self-cleaving peptides; sfGFP, superfolder green fluorescent protein; ZFN, zinc-finger nuclease. Construct not shown to scale.(D) RT-qPCR analysis of target gene expression after CRISPRi of TFRC (TFRCi) or CRISPRa of MYOD1 (MYOD1a), compared to matched controls (NTCi/a). cs1/2, 10X Genomics capture sequences 1/2; NTC, non-targeting control; K, KRAB; PH, p65-HSF1. N = 2–9 clones; two-way ANOVA with protospacer type and guide::effector architecture as factors, followed by Holm–Sidak’s multiple-comparison test for targeting versus NTC comparisons within each architecture (asterisks), and all pairwise architecture comparisons within the same guide type (daggers). *, † p < 0.05; **, †† p < 0.01; ***, ††† p < 0.001; p > 0.05 not reported.(E) Schematic of the selected orthogonal CRISPRa and CRISPRi modules. MS2-cs2 sgRNAs recruit MCP-PH for activation, whereas com-cs1 sgRNAs recruit Com-KRAB for repression, enabling distinct effectors to be recruited to different loci from the same dCas9 chassis. Note that MCP binds internal MS2 hairpins as a dimer, whereas Com binds as a monomer and requires the 3’-terminal com hairpin context, including the base of the stem.
Fig 2: Validation of inducible CRISPRa/i in hiPSCs and cardiac organoids(A) RT-qPCR validation of inducible TFRC CRISPRi in a clonal TetCas hiPSC line re-targeted at AAVS1 with the CRISPRi architecture optimized in Figure 1. The clone was homozygously edited and lacked random integrations. Cells were treated with Tet (+) for the indicated times and compared with matched untreated controls (−). N = 2 cultures; two-way ANOVA with Tet treatment and time as factors followed by Holm-Sidak’s multiple-comparison test for − versus +. Here and throughout the figure, * = p < 0.05; ** = p < 0.01; *** = p < 0.001.(B) As in panel A, but for inducible SOX2 and NANOG CRISPRi after 3 days of Tet treatment, with Tet-treated NTC controls shown where applicable. N = 2–4 clones; one-sample two-tailed t-tests against the indicated pooled Tet-untreated reference values (black dotted lines).(C) As in panel A, but for inducible MYOD1 CRISPRa after 5 days of Tet treatment, compared with matched untreated and NTC controls. The pink dotted line indicates the benchmark level of MYOD1 expression achieved by architecture-matched constitutive CRISPRa in Cas-R hiPSCs (Figure 1D). N = 3 clones; one-sample two-tailed t-test against the indicated pooled Tet-untreated MYOD1 CRISPRa reference value (black dotted line).(D) As in panel A, but for inducible ASCL1 and HNF1A CRISPRa after 3 days of Tet treatment. N = 3 clones; one-sample two-tailed t-tests against the indicated pooled Tet-untreated reference values (black dotted lines).(E) Representative widefield fluorescence images of day 7.5 cardiac organoids (cardioids) generated from TFRC CRISPRi hiPSCs, showing maintenance of dCas9–2A-mCherry and Com-K-2A-sfGFP. Scale bar, 800 μm.(F) As in panel A, but for inducible TFRC CRISPRi in day 7.5 cardiac organoids Tet-treated for 3 days. N = 3 clones; one-sample two-tailed t-test against the indicated Tet-treated NTC reference value (black dotted line).(G) As in panel A, but for inducible MYOD1 and ASCL1 CRISPRa, together with NTC controls, in day 7.5 cardiac organoids. Organoids were Tet-treated for 24 h and sorted into TTN-mEGFP+ cardiomyocytes and TTN-mEGFP− non-cardiomyocytes before analysis. N = 2 differentiations using 1–2 clones per target; one-sample two-tailed t-tests against the indicated pooled Tet-untreated, TTN-mEGFP-matched reference values (black dotted lines).(H) Schematic of the MYOD1 regulatory regions and sgRNA combinations tested for multiplex CRISPRa. Individual sgRNAs are indicated as A–D. 2xP, two promoter guides; 3xP, three promoter guides; 2xP_1xDRR, two promoter guides plus one distal regulatory region (DRR) guide; PRR, proximal regulatory region, not tested.(I) RT-qPCR validation of multiplex inducible MYOD1 CRISPRa after 24 h of Tet treatment. The three-guide NTC control (3xNTC) and the single-sgRNA benchmark from panel C (yellow dotted line) are shown for comparison. N = 3 clone pools; one-way ANOVA followed by Holm–Sidak’s multiple-comparison test for all pairwise comparisons.(J) Representative phase-contrast images of hiPSCs carrying the 3xP inducible MYOD1 CRISPRa system after 72 h of Tet treatment, compared to Tet-untreated controls. Scale bar, 50 μm.
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