grna sequences  (Addgene inc)

Journal: bioRxiv

Article Title: Lmna deficiency promotes EPHX2 nuclear translocation to ameliorate cardiac dysfunction in mice

doi: 10.64898/2026.02.09.704693

Figure Lengend Snippet: A and B , Immunofluorescence imaging and quantification of P14 isolated cardiomyocytes ( A ) and E13.5 mouse embryonic fibroblasts ( B ). C , Western blot images and quantification of nuclear/cytoplasmic EPHX2 in Lmna Δ/Δ mouse hearts. D , Quantification by qRT-PCR and western blot images of Lmna Δ/Δ hearts. E , Immunofluorescence images and quantification of EPHX2 in isolated cardiomyocytes of P14 CKO mice. F , Immunofluorescence images and quantification of NE rupture and EPHX2 location. P values were generated by Mann-Whitney U test. * P <0.05, ** P <0.01, *** P <0.001, mean ± SD. FC, fold change. n represents numbers of cells ( A , B , E and F ) or mice ( C and D ). LB1, lamin-B1. HA, hemagglutinin. LSL, loxP-stop-loxP. NE, nuclear envelop.

Article Snippet: Ephx2 sgRNA sequences were subcloned to AAV-U6-sgRNA-Scaffold- Tnnt2 -SaCas9-HA-OLLAS (Addgene #209781)[ ] to generate the AAV-U6-sgRNA- Tnnt2 -Sacas9-HA vectors.

Techniques: Immunofluorescence, Imaging, Isolation, Western Blot, Quantitative RT-PCR, Generated, MANN-WHITNEY

Journal: bioRxiv

Article Title: Lmna deficiency promotes EPHX2 nuclear translocation to ameliorate cardiac dysfunction in mice

doi: 10.64898/2026.02.09.704693

Figure Lengend Snippet: A , Diagram of AAV vectors and the workflow of knocking out Ephx2 . AAV was injected subcutaneously. B , Editing efficiency of Ephx2 by amplicon sequencing analysis in hearts. C , Western blot analysis and quantification of AAV-treated cardiac tissues. D , Echocardiography of cardiac function and dimensions at 2 or 3 weeks after AAV treatment. E , Survival curve with the log-rank test between the CKO and CKO+AAV groups, * P <0.05. F , EET quantification of cardiac tissues by enzyme linked immunosorbent assay (ELISA) and mass-spectrometry analysis. P values were generated by Mann-Whitney U test. * P <0.05, ** P <0.01, *** P <0.001, mean ± SD. n represents numbers of mice. FS, fraction shortening. LVIDd, left ventricular internal dimension in diastole. LVIDs, left ventricular internal dimension in systole. EET, epoxyeicosatrienoic acid.

Article Snippet: Ephx2 sgRNA sequences were subcloned to AAV-U6-sgRNA-Scaffold- Tnnt2 -SaCas9-HA-OLLAS (Addgene #209781)[ ] to generate the AAV-U6-sgRNA- Tnnt2 -Sacas9-HA vectors.

Techniques: Injection, Amplification, Sequencing, Western Blot, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Generated, MANN-WHITNEY

Journal: bioRxiv

Article Title: Lmna deficiency promotes EPHX2 nuclear translocation to ameliorate cardiac dysfunction in mice

doi: 10.64898/2026.02.09.704693

Figure Lengend Snippet: A , Diagram of AAV vectors and the workflow of overexpressing nuclear EPHX2. AAV was injected subcutaneously at P1. B , Western blot analysis of EPHX2 overexpression. C , Immunofluorescence images show the expression and localization of EPHX2 in the myocardium of P14 mouse hearts. The yellow arrow points to the nucleus. D , Echocardiography of cardiac function and structure. E , Total DHET/EET in hearts by ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS)-based lipidomic. 6 animals per group. The Kruskal-Wallis H test was used. ns, not significant. F , Schematic diagram of inactivating mutation sites in EPHX2 hydrolase. G , Work Flow for the detection of EPHX2 hydrolase activity. H , Dynamic curve of EPHX2 hydrolysis product concentration. I , Schematic diagram of AAV vectors and experimental workflow (left). Echocardiographic analysis of cardiac function in CKO mice (right). CKO mice were injected with 2×10 11 AAV9 to specifically overexpress hydrolase-deficient EPHX2 in cardiomyocyte nuclei at P1. Echocardiographic analysis was performed at P14. P values were generated by Mann-Whitney U test. * P <0.05, *** P <0.001, mean ± SD. n represents numbers of mice. HA, hemagglutinin. NLS, nuclear localization sequence. NES, nuclear export sequence. t-AUCB, trans-4-(4-[3-Adamantan-1-yl-ureido]-cyclohexyloxy)-benzoic acid, a small-molecule inhibitor of EPHX2 hydrolase activity. FS, fraction shortening. LVIDd, left ventricular internal dimension in diastole. LVIDs, left ventricular internal dimension in systole.

Article Snippet: Ephx2 sgRNA sequences were subcloned to AAV-U6-sgRNA-Scaffold- Tnnt2 -SaCas9-HA-OLLAS (Addgene #209781)[ ] to generate the AAV-U6-sgRNA- Tnnt2 -Sacas9-HA vectors.

Techniques: Injection, Western Blot, Over Expression, Immunofluorescence, Expressing, Liquid Chromatography, Mass Spectrometry, Tandem Mass Spectroscopy, Mutagenesis, Activity Assay, Concentration Assay, Generated, MANN-WHITNEY, Sequencing

Journal: bioRxiv

Article Title: Lmna deficiency promotes EPHX2 nuclear translocation to ameliorate cardiac dysfunction in mice

doi: 10.64898/2026.02.09.704693

Figure Lengend Snippet: A , Diagram of gene therapy of NLS knock-in via HDR. B , Work flow of the AAV-HDR system. C , Example of targeted cDNA amplicon-sequencing results. The edited genomic regions were analyzed using CRISPResso2 software, and the editing efficiencies of HDR and NHEJ were calculated separately. D , AAV dose-dependent editing efficiency in mouse hearts evaluated by amplicon sequencing. E , Immunofluorescence images and quantification of EPHX2-positive nucleus in myocardium. n=3. F , Western blot images and quantification of nuclear EPHX2. n=3. G , Diagram of AAV-HDR and control AAVs, and outcomes in Lmna F/F ; Rosa CAG-LSL-Cas9-tdGFP mice after treatment. H , Editing efficiency of HDR and NHEJ-mediated gene edting after 4×10 11 AAV treatment by amplicon sequencing. n=4. I , Echocardiogram analysis of AAV-HDR treated hearts. J , Western blot images and quantification of γH2AX in myocardium. P values were generated by Mann-Whitney U test. * P <0.05, ** P < 0.01, *** P < 0.001, mean ± SD. n represents numbers of mice. HDR, homology-directed repair. NHEJ, non-homologous end-joining. HA, homology arm. Cre, Cyclization recombination. LSL, loxP-stop-loxP. NLS, nuclear localization signal. KO, knock-out. KI, knock-in. FS, fraction shortening. LVIDd, left ventricular internal dimension in diastole. LVIDs, left ventricular internal dimension in systole.

Article Snippet: Ephx2 sgRNA sequences were subcloned to AAV-U6-sgRNA-Scaffold- Tnnt2 -SaCas9-HA-OLLAS (Addgene #209781)[ ] to generate the AAV-U6-sgRNA- Tnnt2 -Sacas9-HA vectors.

Techniques: Knock-In, Amplification, Sequencing, Software, Immunofluorescence, Western Blot, Control, Generated, MANN-WHITNEY, Non-Homologous End Joining, Knock-Out