Journal: bioRxiv

Article Title: Virus-free continuous directed evolution in human cells using somatic hypermutation

doi: 10.1101/2024.12.24.629435

Figure Lengend Snippet: ( A ) Transfection scheme for RA 1 cells using a two-plasmid system. ( B ) Integration scheme of homology cassette, following DSB formation by RNA guided Cas9. ( C ) Selection scheme for transfected RA 1 cells containing the puromycin resistance cassette, puroR. ( D ) FACS density plot of gated eGFP+ cells in untransfected RA 1 cells and those enriched through puromycin selection; FL1 represents the FITC fluorescence channel. ( E ) Imaging performed on enriched RA 1-274-eGFP cells following puromycin selection. Scale bar indicates 100 µM.

Article Snippet: The integration plasmid, p274, was sourced from Addgene (ID 164851), and the Cas9/sgRNA plasmid, p276, was also sourced from Addgene (ID 164850).

Techniques: Transfection, Plasmid Preparation, Selection, Fluorescence, Imaging

Journal: Science translational medicine

Article Title: Precise genomic editing of pathogenic mutations in RBM20 rescues dilated cardiomyopathy

doi: 10.1126/scitranslmed.ade1633

Figure Lengend Snippet: (A) ABE correction of the R636Q mutation using sgRNA (blue) and ABEmax-VRQR-SpCas9. On-target: A6 (red). Bystander: A14 and A20 (green). Silent: A4, A13, and A19 (brown). (B) Percentage of adenine-to-guanine editing determined by deep sequencing of cDNA from hearts of R636Q/R636Q mice (6 weeks after ABE correction). Data are expressed as means ± SEM (n = 3). (C) Fractional shortening at 4 and 8 weeks after ABE correction in WT, R636Q/+, R636Q/R636Q, and corrected mice. Data are expressed as means ± SEM (n = 6 per group). Two-way ANOVA with Tukey’s multiple comparisons test was performed. ****P < 0.0001. (D) H&E staining of four-chamber hearts (12 weeks after ABE correction). Scale bar, 1 mm. LV, left ventricle. (E) Kaplan-Meier survival curve of all groups (n = 16 per group). Log-rank (Mantel-Cox) test was performed. ****P < 0.0001 for R636Q/R636Q versus other groups. (F) Immunohistochemistry showing the translocation of RBM20 in CMs of WT, R636Q/+, R636Q/R636Q, and corrected mice (12 weeks after ABE correction). cTnT (red), RBM20 (green), and DAPI (blue). Scale bar, 10 μm. (G) Quantification of RBM20 localization before and after correction in R636Q/R636Q mice. Data are expressed as means ± SEM (n = 4 per genotype). Two-way ANOVA with Bonferroni’s multiple comparisons test was performed. ****P < 0.0001.

Article Snippet: The N-terminal and C-terminal regions of ABEmax-VRQR-SpCas9 were extracted from CMV_Npu-ABEmax N-terminal (Addgene plasmid no. 137173) ( 52 ) and hu6 HGPS sgRNA expression and ABE7.10max VRQR C-terminal AAV vectors (Addgene plasmid no. 154430) ( 28 ) from D. Liu’s laboratory, respectively.

Techniques: Mutagenesis, Sequencing, Staining, Immunohistochemistry, Translocation Assay

Journal: Cell reports

Article Title: SMYD5 is a regulator of the mild hypothermia response

doi: 10.1016/j.celrep.2024.114554

Figure Lengend Snippet: (A) Overexpressed FLAG-tagged SMYD5 ( n = 2) in mESCs binds at promoters of Sp1 and Cirbp but not of Rbm3 . H3K36me3 peaks over Sp1 , Cirbp , and Rbm3 promoter and gene body regions. Data (GEO: GSE184894) are from Zhang et al. (B) Venn graph showing both up- and downregulated SMYD5-bound genes in an RNA-seq of Smyd5 KO cells ( n = 2). Statistical analysis was done with the GeneOverlap package in R using Fisher’s exact test. (C) Smyd5 KO leads to increased mRNA expression of SP1 but not RBM3 or CIRBP in mESCs. (D) SMYD5 knock down (KD) by siRNA yields higher levels of fluorescence of SP1-MHI at 32°C and 37°C compared to the empty vector control. Each data point is a biological replicate ( n = 3) that has been normalized against the same non-transfected HEK293WT+Cas9+SP1-MHI cell line; mean and SD are depicted where applicable. Significance levels were calculated with Šidák’s multiple-comparisons test in GraphPad Prism. (E) Relative expression of SMYD5 mRNA compared to GAPDH from SMYD5 KO cells, measured by RT-qPCR at 37°C, with or without rescue with the FLAG-SMYD5 sgRNAres plasmid (labeled SMYD5 sgRNA#6res). Each data point is a biological replicate ( n = 4), with mean and SD where applicable. Significance levels were calculated with an unpaired one-tailed t test in GraphPad Prism. (F) Western blot using antibodies against SMYD5 and Lamin B in an SMYD5 KO HEK293 cell line at 37°C with and without rescue with the FLAG-SMYD5 sgRNAres plasmid (labeled SMYD5 sgRNA#6res). SMYD5 KO was successful at the protein level at 37°C ( n = 2–3). Data are shown as in (E). (G) Relative expression of SP1 mRNA compared to GAPDH from SMYD5 KO cells, measured by RT-qPCR at 37°C, with or without rescue with the FLAG-SMYD5 sgRNAres plasmid (labeled SMYD5 sgRNA#6res) ( n = 4). Data are shown as in (E). (H–J) Western blot quantification with and without 16-h incubation at 32°C and representative examples using antibodies against SP1, CIRBP, and RBM3, respectively, in SMYD5 KO cells. Each data point is a biological replicate ( n = 2–3), with mean and SD where applicable. Significance levels were calculated with an unpaired one-tailed t test in GraphPad Prism. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Two ready-made sgRNA vectors (GenScript, SMYD5 plasmid vector #2 and #6) were packed into lentivirus according to the protocol described by Kutner et al., using pMD2.G (Addgene, #12259) and psPAX2 (Addgene, #12260).

Techniques: RNA Sequencing, Expressing, Knockdown, Fluorescence, Plasmid Preparation, Control, Transfection, Quantitative RT-PCR, Labeling, One-tailed Test, Western Blot, Incubation