Journal: BMC genomics

Article Title: Optimal SpCas9- and SaCas9-mediated gene editing by enhancing gRNA transcript levels through scaffold poly-T tract reduction.

doi: 10.1186/s12864-025-11317-2

Figure Lengend Snippet: Fig. 2 The modified 3TC scaffold boosts SpCas9 gRNA expression levels compared to the original 4T scaffold. (A) DNA sequence of the 4T and modified 3TC scaffolds. (B) Relative quantification (RQ) of mDmd gRNA delivered by nucleofection of PX459.V2 (4T) to C2C12 cells, by qRT-PCR. Mean Log2RQ ± 95% CI; n = 3. One-way ANOVA with Dunnett’s multiple comparisons test was performed on ΔΔCT values, **p ≤ 0.01, ***p ≤ 0.001. (C, D, E) Comparison of the relative quantities of mDmd Sp gRNA, delivered by PX459.V2, pdg459.V2 (2 × 4T) and PX459.V3 (3TC), measured by qRT-PCR. Mean Log2RQ ± 95% CI; n = 3. One-way ANOVA with Tukey’s multiple comparisons test performed on ΔΔCT values, **p ≤ 0.01, ***p ≤ 0.001

Article Snippet: SaCas9Puro.V2 was generated by replacing the SpCas9 coding sequences of PX459.V2 with SaCas9 coding sequences from PX601 (Addgene #61591).

Techniques: Modification, Expressing, Sequencing, Quantitative Proteomics, Quantitative RT-PCR, Comparison

Journal: BMC genomics

Article Title: Optimal SpCas9- and SaCas9-mediated gene editing by enhancing gRNA transcript levels through scaffold poly-T tract reduction.

doi: 10.1186/s12864-025-11317-2

Figure Lengend Snippet: Fig. 5 Editing efficiencies of high-fidelity SpCas9s with the 3TC scaffold. Comparison of PX459.V2 SpCas9-HF1 (4T), PX459.V3 SpCas9-HF1 (3TC), PX459.V2 eSpCas9(1.1) (4T) and PX459.V3 eSpCas9(1.1) (3TC) plasmids de livered by lipofection at a (A) high and (B) low plasmid dose without puro mycin selection in HEK239T cells, assessed by deep amplicon sequencing. Mean ± SEM; n = 3. Two-way ANOVA with Šídák’s multiple comparisons test; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (C) Editing efficiencies of hDMD-B in the G19 gRNA configuration with WT and high-fidelity Sp-Cas9 plasmids delivered by nucleofection with puromycin selection in HEK293Ts. Two- way ANOVA with Šídák’s multiple comparisons test; *p ≤ 0.05, ***p ≤ 0.001

Article Snippet: SaCas9Puro.V2 was generated by replacing the SpCas9 coding sequences of PX459.V2 with SaCas9 coding sequences from PX601 (Addgene #61591).

Techniques: Comparison, Plasmid Preparation, Selection, Amplification, Sequencing

Journal: Molecules and Cells

Article Title: RPS5A Promoter-Driven Cas9 Produces Heritable Virus-Induced Genome Editing in Nicotiana attenuata

doi: 10.14348/molcells.2021.0237

Figure Lengend Snippet: A. tumefaciens containing TRV2 derivatives was mixed with Agrobacterium carrying TRV1 in a ratio of 1:1. The mixed solution was co-infiltrated into the leaves of 4- to 5-week-old SpCas9-producing N. attenuata . M 1 seeds were collected from TRV-infected plants and germinated. Equal amounts of leaf tissues from M 1 seedlings (n = 11-26) were pooled for performing targeted deep sequencing. The editing frequencies in individual plants of groups harboring a mutant were examined. It takes three to four months to obtain gene-edited M 1 seeds. nos, nopaline synthase terminator; SGP, TRV subgenomic promoter; CP, coat protein; pSGP, pea early-browning virus subgenomic promoter; LB, left border; RB, right border.

Article Snippet: The coding sequence of SpCas9 was synthesized by Macrogen (Korea) and amplified with Cas9-F and Cas9-R primers.

Techniques: Infection, Sequencing, Mutagenesis, Virus