Journal: Nature Communications

Article Title: Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases

doi: 10.1038/s41467-021-24454-5

Figure Lengend Snippet: a Liver editing in mouse using sRGN-LNPs and SpyCas9-LNPs at doses of 1.5 and 2 mg/kg. LNPs or PBS control was administered intravenously. Data are presented as mean ± SD, each dot represents an independent biological replicate; sRGN3.3: n = 8 at 1.5 mg/kg and n = 4 at 2 mg/kg, sRGN3.1: n = 8 at both doses, SpyCas9: n = 7 at 1.5 mg/kg and n = 8 at 2 mg/kg. All mRNA constructs were m1Ψ base-substitution modified. All mRNAs used in this work were of comparable quality: full-length purity ≥85% and low dsRNA levels effected by reverse-phase purification. sRGN3.3 and sRGN3.1 used the same end-modified sgRNA against albumin ( Alb -T1), while SpyCas9 used a similar sgRNA with a protospacer shifted by one nucleotide (to accommodate “NGG” PAM instead of “NNGG”, sequences in Supplementary Table ) and internal 2′O-methyl modifications (see Supplementary Fig. ). Significance was tested using the Kruskal–Wallis test (alpha = 0.05) corrected for multiple comparisons using Dunn’s test; * p < 0.05; ** p < 0.01; ns = not significant (1.5 mg/kg: sRGN3.3 vs 3.1: p = 0.0027, sRGN3.1 vs Spy: p > 0.9999, sRGN3.3 vs Spy: p = 0.0053; 2 mg/kg: sRGN3.3 vs 3.1: p = 0.0449, sRGN3.1 vs Spy: p > 0.9999, sRGN3.3 vs Spy: p = 0.0780). b top: schematic of the genetic construct, packaging, and delivery of AAV5 vector. Bottom: in vivo editing of nonhuman primate (NHP) photoreceptors by subretinal injection of rAAV5 vectors. AAV5 vectors carrying sRGN3.1 and WT-IVS40 sgRNA (guide_111) were injected into the subretinal space of NHPs. Indels were quantified by amplicon sequencing from retinal punches. Individual measurements and bar as mean, n = 3 eyes at 6 weeks and n = 4 eyes at 12 weeks, independent biological replicates. A previous study and our internal assessment demonstrated that nucleases driven by a photoreceptor-specific GRK1 promoter are expressed only by photoreceptors, which account for approximately 30% of the cells in retinal punches , . We therefore calculated the frequency of indels in the photoreceptor fraction with a multiplier of 3.3. Source data are provided in the source data file.

Article Snippet: For off-target analyses on the USH2A (IVS40) locus, the homozygous IVS40 293FT cell line (2 × 10 5 cells) was nucleofected with 5 pmol of dsODN and 2 μg of a plasmid carrying sRGN3.1 and T428 sgRNA in Lonza SF buffer by nucleofection under Program CM-130.

Techniques: Control, Construct, Modification, Purification, Plasmid Preparation, In Vivo, Injection, Amplification, Sequencing

Journal: Scientific Reports

Article Title: Aquaporin7 plays a crucial role in tolerance to hyperosmotic stress and in the survival of oocytes during cryopreservation

doi: 10.1038/srep17741

Figure Lengend Snippet: ( A ) Immunofluorescence analysis of AQP7 expression in mouse oocytes in the presence of 0.25 M, 0.5 M, 0.75 M and 1 M sucrose, respectively. ( B ) Summary data of the immunofluorescence analysis (n ≥ 5). ( C ) Immunofluorescence analysis of AQP7 and F-actin colocalization in mouse oocytes (red: AQP7; green: F-actin). Scale bar ( A – C ), 20 μm, ( D ) Co-immunoprecipitation of AQP7 and F-actin. IP, immunoprecipitation. ( E ) Green fluorescent protein (GFP) intensity in 293FT cells transfected with the GFP-hAQP7 fusion protein expression plasmid or with the GFP vector alone in the presence of 8% EG, 9.5% DMSO, and 0.5 M sucrose, respectively. Scale bar, 10 μm. ( F ) Summary data of the immunofluorescence analysis (calculated for 20 cells for each condition obtained from three independent experiments). Data are presented as the mean ± SE, ** P < 0.01 compared to the corresponding control.

Article Snippet: The human embryonic kidney cell line (293FT) was purchased from ScienCell (Carlsbad, USA), and cultured in DMEM (Gibco, Grand Island, USA) containing 10% fetal bovine serum (Gibco).

Techniques: Immunofluorescence, Expressing, Immunoprecipitation, Transfection, Plasmid Preparation, Control

Journal: Scientific Reports

Article Title: Aquaporin7 plays a crucial role in tolerance to hyperosmotic stress and in the survival of oocytes during cryopreservation

doi: 10.1038/srep17741

Figure Lengend Snippet: ( A ) Oocytes were pretreated with LY294002, staurosporine, U0126 and SP600125, respectively, and the immunofluorescence intensities of AQP7 were analysed in mouse oocytes in the presence of 8% EG. Scale bar, 20 μm. ( B ) Summary data of the immunofluorescence analysis (n ≥ 7). ( C ) 293FT cells were transfected with the GFP-hAQP7 fusion protein expression plasmid and treated as in ( A ). The immunofluorescence intensities of GFP-hAQP7 were analysed in the presence of 8% EG. Scale bar, 10 μm. ( D ) Summary data of the immunofluorescence analysis (calculated for 20 cells for each condition obtained from three independent experiments). ( E ) Western blotting analysis of GFP-hAQP7 in 293FT cells treated as in ( C ). ( F ) Summary data of the Western blotting analysis (n = 3). Data are presented as the mean ± SE. ** P < 0.01 compared to the corresponding control.

Article Snippet: The human embryonic kidney cell line (293FT) was purchased from ScienCell (Carlsbad, USA), and cultured in DMEM (Gibco, Grand Island, USA) containing 10% fetal bovine serum (Gibco).

Techniques: Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Western Blot, Control