Journal: Scientific Reports

Article Title: Targeted mutagenesis in soybean using the CRISPR-Cas9 system

doi: 10.1038/srep10342

Figure Lengend Snippet: Construction of binary vectors for genome editing in soybean. Cas9 fused with a single nuclear localization signal (NLS) is expressed with a Cauliflower mosaic virus 35s (CaMV 35s) promoter. Synthetic guide RNA (sgRNA) is derived using U6 promoters. ( a ) Arabidopsis thaliana U6-26 promoter ( b ) Glycine max U6-10 promoter. Sequences containing two Bsa I sites are located between the U6 promoter and the sgRNA scaffold. These sequences can be easily replaced with a gene-specific sgRNA seed. LB: left border; RB: right border.

Article Snippet: Arabidopsis U6-26 and soybean U6-10 promoters with sgRNA were synthesized (Genscript, Nanjing, China) ( and ) and cloned into pUC57-Kan vectors to generate pUC57-AtU6-26-sgRNA and pUC57-GmU6-10-sgRNA plasmids, respectively.

Techniques: Virus, Derivative Assay

Journal: Frontiers in Genome Editing

Article Title: Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane

doi: 10.3389/fgeed.2021.654996

Figure Lengend Snippet: Strategy for targeted mutagenesis of sugarcane MgCh and confirmation of In-vitro cleavage activity of sgRNAs. (A) Schematic representation of sugarcane MgCh locus and sgRNAs' target sites, sgRNA1 targeting nts 731–750 and sgRNA2 targeting nts 1223–1242. Mutations at sgRNA target site 1 would disrupt the Nco I restriction recognition site. Exons are indicated with green boxes, and introns with yellow boxes. (B) In vitro cleavage assay to validate sgRNA activity, the (810 nts) MgCh PCR amplicon is digested by ribonucleoprotein complex (RNP) of Cas9 and either sgRNA1 or sgRNA2 into ~581 and 229 nts or 701 and 109 nts, respectively. (C) Map of sugarcane gene editing plasmid (pMGE); Two sgRNAs are monoscistronically expressed under Oryza sativa U6 promoter, npt II is under transcriptional control of cauliflower mosaic virus (CaMV) 35S promoter and CaMV terminator, Cas9 is under transcriptional control of CaMV 35S promoter and Sorghum bicolor HSP18 terminator. Protospacer adjacent motifs (PAMs) are indicated in red font.

Article Snippet: sgRNA vectors containing Oryza sativa U6 promoter were designed and custom synthesized in the pUC57 backbone (Genscript, NJ, USA) to generate pUCMg12.

Techniques: Mutagenesis, In Vitro, Activity Assay, Cleavage Assay, Amplification, Plasmid Preparation, Control, Virus

Journal: Frontiers in Molecular Neuroscience

Article Title: The Generation of Mouse and Human Huntington Disease iPS Cells Suitable for In vitro Studies on Huntingtin Function

doi: 10.3389/fnmol.2017.00253

Figure Lengend Snippet: The silencing cassette design and derivation of the iPSC lines. (A) Schematic of the psiOrange vector. The silencing cassette is inserted into the iPSC genome as a piggyBac (PB) transposon bordered by 5′ and 3′ PB arms. An shRNA is expressed under regulation of a U6 promoter and is flanked by pri-miR-30 5′ and 3′ sequences, which are 151 and 128 bp long, respectively. Additionally, the mOrange2 fluorescent reporter and NeoR resistance genes are included within the transposon. (B) Schematic of shRNA sequences. Effector guide strands are marked in red. (C) mOrange reporter expression is sustained in the mouse iPSC state and upon differentiation as embryonic bodies (EB) or neuroectoderm (N2B27 conditions). (D) mOrange reporter is expressed in human iPSCs with shRNA cassettes.

Article Snippet: Constructs (Figure ) composed of a U6 promoter, a miR-30 5′ flank (151 bp), an shRNA sequence, a miR-30 3′ flank (128 bp), a U6 terminator (TTTTTT), an EF1alpha promoter, an mOrange2 reporter gene, and an SV40 pA site were were synthesized by Genscript (Piscataway, NJ) and cloned into a pPB-HKS-neoL vector obtained, by removing the EGFP reporter gene, from a pPB-UbC.eGFP-neo plasmid (Yusa et al., ).

Techniques: Plasmid Preparation, shRNA, Expressing