expression vectors pet28a cas9 his  (Addgene inc)

Journal: Journal of Nanobiotechnology

Article Title: Finely tuned ionizable lipid nanoparticles for CRISPR/Cas9 ribonucleoprotein delivery and gene editing

doi: 10.1186/s12951-024-02427-2

Figure Lengend Snippet: Schematic on the preparation of CrLNP for CRISPR/Cas9 RNP delivery. CrLNPs were prepared using ionizable lipids and precise control of the pH conditions, to enable efficient delivery and gene editing in target cells

Article Snippet: Cas9 expression plasmid pET28a-Cas9-His ( Addgene, Additional file 1: Figure A) was transformed to E.coli Rosetta (DE)3 cells using the heat shock method.

Techniques: CRISPR, Control

Journal: Journal of Nanobiotechnology

Article Title: Finely tuned ionizable lipid nanoparticles for CRISPR/Cas9 ribonucleoprotein delivery and gene editing

doi: 10.1186/s12951-024-02427-2

Figure Lengend Snippet: Characterization of CrLNPs. A Cleavage activities of Cas9 stored at various pHs, by adding the RNP with target DNA (target: EGFR ). B Hydrodynamic size, C zeta potential, and D cryo-TEM of CrLNPs prepared at different pHs (pH 6.0 and 7.4). Scale bar, 100 nm. E TNS assay of CrLNP prepared at pH 6.0. F Loading efficiency of Cas9 and sgRNA in CrLNPs prepared at different pHs (pH 6.0 and 7.4)

Article Snippet: Cas9 expression plasmid pET28a-Cas9-His ( Addgene, Additional file 1: Figure A) was transformed to E.coli Rosetta (DE)3 cells using the heat shock method.

Techniques: Zeta Potential Analyzer

Journal: Journal of Nanobiotechnology

Article Title: Finely tuned ionizable lipid nanoparticles for CRISPR/Cas9 ribonucleoprotein delivery and gene editing

doi: 10.1186/s12951-024-02427-2

Figure Lengend Snippet: Validation and optimization CrLNP 6.0 on gene editing in vitro. Gene editing efficiencies by treating cells with CrLNPs targeting the surrogate reporter gene under various A lipid compositions, and B N/P ratios. C Scheme of endogenous gene ( IL-10 ) editing in various cell lines analyzed by targeted deep sequencing. Indel frequencies of CT26 cells by treating with CrLNPs prepared at different Cas9 to sgRNA molar ratios (1:1, or 1:3) analyzed by D T7E1 assay and E NGS. F Representative sequence data from E for CrLNP 6.0 (orange: crRNA, blue: PAM, -: deletion, WT: wild-type). G Indel frequencies of various cell lines treated with CrLNP 6.0 at different molar ratios of MC3. H Changes in efficiencies of gene editing in cells treated with CrLNP 6.0 stored at 4℃ for various incubation times. I Indels at possible off-target sites in CT26 cells treated with CrLNP 6.0

Article Snippet: Cas9 expression plasmid pET28a-Cas9-His ( Addgene, Additional file 1: Figure A) was transformed to E.coli Rosetta (DE)3 cells using the heat shock method.

Techniques: Biomarker Discovery, In Vitro, Sequencing, Incubation

Journal: Journal of Nanobiotechnology

Article Title: Finely tuned ionizable lipid nanoparticles for CRISPR/Cas9 ribonucleoprotein delivery and gene editing

doi: 10.1186/s12951-024-02427-2

Figure Lengend Snippet: In vivo delivery of CrLNPs. A Scheme on the treatment of CrLNP 6.0 to HEK293T xenograft mouse model. CrLNP 6.0 , including AF647-conjugated Cas9, was treated for fluorescence imaging. B IVIS imaging at different time points after intratumoral injection of CrLNP 6.0 , and C quantification of signals ( n = 3). Data are shown as mean ± SD, * P < 0.05, **** P < 0.0001 by student’s t -test. D Confocal microscopy of tumor tissues 72 h after injection of CrLNP 6.0 . Scale bar, 40 μm. Flow cytometry of cells from tumor tissues 72 h after injection with CrLNP 6.0 , shown by E scatter plot and F histogram of representative samples for each group, as well as G mean values for each group ( n = 3). Data are shown as mean ± SD, * P < 0.05 by one-way ANOVA.

Article Snippet: Cas9 expression plasmid pET28a-Cas9-His ( Addgene, Additional file 1: Figure A) was transformed to E.coli Rosetta (DE)3 cells using the heat shock method.

Techniques: In Vivo, Fluorescence, Imaging, Injection, Confocal Microscopy, Flow Cytometry