Journal: International Journal of Molecular Sciences

Article Title: Suppression of SHROOM1 Improves In Vitro and In Vivo Gene Integration by Promoting Homology-Directed Repair

doi: 10.3390/ijms21165821

Figure Lengend Snippet: Paired crRNA library screening identified that SHROOM1 is an HDR suppressor of dual-cut BFP (blue fluorescence protein) reporter. ( a ) Schematic of paired crRNA library screening strategy in the main text; ( b ) multiple genes were enriched in the HDR population. Representative genes are highlighted in red. Data were generated from n = 4 independent experiments. The p -value was calculated using a two-sided Student’s t -test. ( c ) Relative HDR ratio of dual-cut BFP reporter cells treated with individual siRNA of top eight genes enriched in the screening. Data were generated from n = 3 independent experiments. Error bars, ± SD * p < 0.05; ** p < 0.01; *** p < 0.001 by two-sided Student’s t -test. ( d ) Relative HDR ratio-time curve of dual-cut BFP reporter cell lines expressing mono-paired crRNAs. Data were collected every two days from day 20 to day 36 after cell sorting. Data were generated from n = 3 independent experiments. ( e ) Genes enriched in the HDR population of simulated screening. Data were generated from n = 4 independent experiments. Error bars, ±SD * p < 0.05; ** p < 0.01; *** p < 0.001 by two-sided Student’s t -test.

Article Snippet: Primary antibodies against the following proteins were used: SHROOM1 (bs-13735R; Bioss, Beijing, China); GAPDH (sc-365062; Santa Cruz, Dallas, TX, USA).

Techniques: Library Screening, Fluorescence, Generated, Expressing, FACS

Journal: International Journal of Molecular Sciences

Article Title: Suppression of SHROOM1 Improves In Vitro and In Vivo Gene Integration by Promoting Homology-Directed Repair

doi: 10.3390/ijms21165821

Figure Lengend Snippet: Knockdown of SHROOM1 enhances the knock-in efficiency of cells in vitro. ( a ) Schematic overview of gene-targeting strategies with siRNA and different types of donor at the FBL locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; IF/IR, inserted forward/reverse primer. ss, single strand; ds, double strand; dc, double cut. ( b ) Experimental scheme for targeted FBL -2A-GFP knock-in in HEK293T cells. Representative visual fields and sorting charts ( c ) and relative knock-in efficiency. Scale bar, 200 μm. ( d ) of ss, ds, and dc donor-based strategies with SHROOM1 siRNA or not at the FBL locus in HEK293T cells. Data were generated from n = 3 independent experiments. Error bars, ± SD ** p < 0.01; *** p < 0.001 by two-sided Student’s t -test ( e , f ). Relative knock-in efficiency of ds and dc donor-based strategies with siRNA in HEK293T, HCT116, or Hepa1-6 cells. Data were generated from n = 3 independent experiments. Error bars, ± SD * p < 0.05; ** p < 0.01 by two-sided Student’s t -test.

Article Snippet: Primary antibodies against the following proteins were used: SHROOM1 (bs-13735R; Bioss, Beijing, China); GAPDH (sc-365062; Santa Cruz, Dallas, TX, USA).

Techniques: Knock-In, In Vitro, Generated

Journal: International Journal of Molecular Sciences

Article Title: Suppression of SHROOM1 Improves In Vitro and In Vivo Gene Integration by Promoting Homology-Directed Repair

doi: 10.3390/ijms21165821

Figure Lengend Snippet: Knockdown of SHROOM1 enhances the knock-in efficiency of mouse embryos. ( a ) Experimental design of micro-injection. Cas9-Avidin mRNA, sgRNA, biotin-ss donor, and siRNA were injected into mouse zygotes and the injected zygotes were transferred to pseudo-pregnant mice for genotyping analysis. Genotyping of Ddx4 locus ( b ) and Icos locus ( c ) in mice treated with SHROOM1 siRNA or NC siRNA after incision by CRISPR/Cas9; ( d ) Summary of SHROOM1 siRNA-mediated HDR at the Ddx4 and Icos loci. Results with significant differences are highlighted in red.

Article Snippet: Primary antibodies against the following proteins were used: SHROOM1 (bs-13735R; Bioss, Beijing, China); GAPDH (sc-365062; Santa Cruz, Dallas, TX, USA).

Techniques: Knock-In, Injection, Avidin-Biotin Assay, CRISPR

Journal: International Journal of Molecular Sciences

Article Title: Suppression of SHROOM1 Improves In Vitro and In Vivo Gene Integration by Promoting Homology-Directed Repair

doi: 10.3390/ijms21165821

Figure Lengend Snippet: SHROOM1 is a potent suppressor of HDR progress. ( a ) Schematic of SHROOM1 deletion using CRISPR/Cas9 and two sgRNAs in HEK293T cells. PAM, highlighted in red; protein codon, highlighted in blue or green, KO for knockout; ( b ) Western blot of different types of cells or treatment. KO, for knockout; p SHROOM1 for SHROOM1 cDNA contained plasmid; relative knock-in efficiency ( c ) and representative visual fields and sorting charts ( d ) in SHROOM1 knockout or wild-type HEK293T cells with treatments. YU238259, an HR inhibitor; Scr7, a NHEJ inhibitor; dc, double-cut sites contained donor. Data were generated from n = 3 independent experiments. Error bars, ± SD *** p < 0.001; n.s., no significance; by two-sided Student’s t -test. Scale bar, 200 μm.

Article Snippet: Primary antibodies against the following proteins were used: SHROOM1 (bs-13735R; Bioss, Beijing, China); GAPDH (sc-365062; Santa Cruz, Dallas, TX, USA).

Techniques: CRISPR, Knock-Out, Western Blot, Plasmid Preparation, Knock-In, Generated