pgcas9 -expressing cassette and u6 promoter: grna backbone  (GenScript corporation)

Journal: Frontiers in Plant Science

Article Title: Efficient Multi-Sites Genome Editing and Plant Regeneration via Somatic Embryogenesis in Picea glauca

doi: 10.3389/fpls.2021.751891

Figure Lengend Snippet: The T-DNA structure of PgCas9 / PaU6 . The hygromycin phosphate transferase ( hpt ) gene, which was driven and terminated by the CaMV35S promoter and the CaMV35S polyA, respectively, was used as the selective marker gene. The PgCas9 with a nuclear localization signal (NLS) at each side of 5′ and 3′ ends was driven by the doubled 35S promoter and terminated by the Nos terminator. The PaU6 promoter was used to drive the expression of polycistronic tRNA-gRNA (PTG), and two Bsa I sites were designed between the PaU6 promoter and the gRNA backbone for assembling the PTG into the binary vector. Eco RI, Sac I, Nco I, Bam HI, Sal I, and Hind III were restriction enzymes. The left and right borders of T-DNA were designated as LB and RB, respectively.

Article Snippet: The final sequence containing PgCas9 -expressing cassette and U6 promoter: gRNA backbone was synthesized by Genscript Biotechnology Co., Ltd (Nanjing, China) and ligated into the modified pCAMBIA1300 vector, in which the Bsa I site was removed with QuickMutation TM (D0206, Beyotime Biotechnology, Shanghai, China), following the manufacturer's introduction.

Techniques: Marker, Expressing, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers

doi: 10.1093/nar/gku836

Figure Lengend Snippet: Activation of the mouse Oct4 and Nanog loci by TALE-As and dCas9-As/gRNAs. ( A ) Schematic diagram of the dCas9-As evaluated in this study. In all cases, blue florescent protein (BFP) was used to track the expression of dCas9-As. gRNA expression is controlled by U6 promoter, and EF1a-mCherry is used to detect the integration of the vector into the genome. A blasticidin resistance and the reverse tetracycline transactivator (rtTA) cassettes were also linked to the mCherry cassette by T2A peptides. PB-5TR and PB-3TR are the two terminal repeat sequences of the piggyBac (PB) transposon. ( B ) Schematic diagram of the TALE and dCas9/gRNA targeting sites at the mouse Oct4 and Nanog enhancers. Red arrows indicate the gRNA targeting sites and the blue arrows mark the TALE sites. ( C ) Activation of the Oct4 distal enhancer luciferase reporter by the TALE-As and dCas9-As/gRNAs. ( D ) qRT-PCR analysis of the Oct4 mRNA levels in MEFs expressing the TALE-As or dCas9-As/gRNAs. ( E ) Activation of the Nanog enhancer luciferase reporter by the TALE-As and dCas9-As/gRNAs. ( F ) qRT-PCR analysis of the Nanog mRNA levels in EpiSCs expressing the TALE-As or dCas9-As/gRNAs. All gene expression values were normalized to Gapdh . Results were representative of three independent experiments and were presented as ±SD, n = 3. * P < 0.05.

Article Snippet: To generate the PB-U6-gRNA–EFα-mCherry-2A-rtTA-2A-BSD vector, the U6 promoter driven gRNA expression cassette (Addgene 44248) was first cloned into the PB-LTR vector.

Techniques: Activation Assay, Expressing, Plasmid Preparation, Luciferase, Quantitative RT-PCR, Gene Expression

Journal: Nucleic Acids Research

Article Title: Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers

doi: 10.1093/nar/gku836

Figure Lengend Snippet: Repression of the Oct4 and Nanog loci by TALE-Rs and dCas9-Rs/gRNAs. ( A ) Schematic diagram of the dCas9-R designs evaluated in this study. In all cases, blue florescent protein (BFP) was used to track the expression of dCas9-R. The gRNA vector used was the same as described in Figure . ( B ) Repression of the endogenous Oct4 locus in Oct4-GFP ES cells indicated by the reduction of GFP intensity in flow cytometric analysis on day 0 and day 3 of expression of the TALE-Rs or dCas9-Rs/gRNAs targeting at the Sites 1–4 of the Oct4 distal enhancer (Gated mCherry + for TALEs and mCherry + /BFP + for dCas9-As/gRNAs). ( C ) Comparison of Oct4 expression levels in Oct4-GFP ES cells expressing the TALE-Rs or dCas9-Rs targeting at the Site 1 or Site 3 of the Oct4 distal enhancer by qRT-PCR. ( D ) The repressive effect of TALE-R and dCas9-Rs/gRNAs targeting at the Site 3 of the Oct4 distal enhancer on MEF reprogramming. MEFs were reprogrammed with Dox inducible CKS and Lrh1 (CKSL) factors together with the TALE-R or dCas9-R/gRNAs as in (C). ‘CKSL+’ indicates that all transfections have CKSL. ‘−’ is the CKSL only control (no repressor). ‘CKS only’ is the reprogramming negative control. ( E ) Reprogramming using FACS-sorted MEFs (as described in 2E) to control transfection variability. Wild-type MEFs were transfected and sorted on day 2 for GFP + /mCherry + in the TALE-R transfection and for GFP + /mCherry + /BFP + in dCas9-R/gRNAs (PL-R) experiments. Both TALE-Rs and dCas9-R/gRNAs (PL-R) targeted the Site 2–4 of the Oct4 distal enhancer. Sorted MEFs were re-plated (20 000 cells/ well) for reprogramming and iPSC colonies were scored by AP staining. ( F ) The repressive effect of TALE-Rs or dCas9-Rs/gRNAs targeting at the Sites 1–2 of the Nanog 5 kb enhancer in Nanog-GFP ES cells. Site 1 is located outside the enhancer region whereas Site 2 is inside. The Nanog repression was demonstrated by the increase of the GFP low/dim fraction in Nanog-GFP ES cells. ( G ) Endogenous N anog mRNA levels in Nanog-GFP ES cells expressing the TALE-Rs or dCas9-Rs targeting at the Sites 1–2. ( H ) Repression of Klf4- mediated EpiSC reprogramming to iPSCs by the TALE-R and dCas9-Rs targeting at the Site 2 of the Nanog 5 kb enhancer. ‘Klf4+’ refers to the transfections combined with Klf4 and ‘−’ is the Klf4 control (no effector). Results were representative of three independent experiments and were presented as ±SD, n = 3.

Article Snippet: To generate the PB-U6-gRNA–EFα-mCherry-2A-rtTA-2A-BSD vector, the U6 promoter driven gRNA expression cassette (Addgene 44248) was first cloned into the PB-LTR vector.

Techniques: Expressing, Plasmid Preparation, Comparison, Quantitative RT-PCR, Transfection, Control, Negative Control, Staining