Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders.

doi: 10.1111/gtc.12379

Figure Lengend Snippet: Figure 4 Genotyping of founders with different phenotypes with RFLP analyses. (A) A schematic of restriction enzyme-RFLP (RE-RFLP) and RNA-guided engineered nuclease-RFLP (RGEN-RFLP) analysis. (B) Estimation of somatic mutation rates of the target alleles in each phenotype. PCR products of tyr and ltk were cleaved by Cas9 protein with sgRNAs in vitro. PCR prod- ucts of slc45a2 were cleaved by BfaI. Cleaved fragments were analyzed by electrophoresis (MultiNA system; Shimadzu). Upper and lower images show Cas9 protein or BfaI (+) and () gel images converted from electropherograms. PCR products of target regions are shown by black arrowheads. Colored arrowheads indicate cleaved products by Cas9 or BfaI. Some extra bands were observed and thought to be large deletions and insertions. The mutation rates were calculated from the molarity of the uncleaved bands (black arrowheads and other extra bands) and the larger cleaved fragments (blue arrowheads). Wt, wild type; W, weak phe- notype; M, moderate phenotype; S, severe phenotype; PS, severe phenotype by Cas9 protein injection; H, half phenotype; F, full phenotype. Full scan images of RFLP analyses are shown in Figs S5–S8 in Supporting Information.

Article Snippet: Preparation and injection of Cas9 and sgRNA Humanized codon hspCas9 cDNA from pX330 (Addgene, #42230; Cong et al. 2013) containing two NLSs was subcloned into a modified pCS2+ vector (pCS2+-T7/hspCas9; Fig. S12 in Supporting Information).

Techniques: Mutagenesis, In Vitro, Electrophoresis, Injection

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders.

doi: 10.1111/gtc.12379

Figure Lengend Snippet: Figure 7 Example of target gene analysis according to our workflow. We designed an sgRNA targeting activin receptor-like kinase 2 (alk2). (A) Representative phenotypes produced by Cas9 mRNA and sgRNA (upper panel). Most phenotypes exhibited a partial loss of ventral fin tissue accompanied by the hypertrophic anus. Normal means no alteration in the ventral fin or anus. The total numbers of individuals are shown at the top of each graph. In vitro fertilization (IVF) indicates control embryos without injection. Frequencies of alk2-disrupted phenotypes from three independent experiments are shown in under panel. (B) RGEN-RFLP analy- sis of somatic mutation rates. (C) Sanger DNA sequencing of somatic mutation alleles from five phenotypes. In total, 107 clones were sequenced. (D) Off-target analysis using heteroduplex mobility assay (HMA). Wt, wild type; P, phenotype.

Article Snippet: Preparation and injection of Cas9 and sgRNA Humanized codon hspCas9 cDNA from pX330 (Addgene, #42230; Cong et al. 2013) containing two NLSs was subcloned into a modified pCS2+ vector (pCS2+-T7/hspCas9; Fig. S12 in Supporting Information).

Techniques: Produced, In Vitro, Control, Injection, Mutagenesis, DNA Sequencing, Clone 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