Customer data for Guide-it products

Introduction                                                                                                                                                                                                                                                                            

Dr. Manabu Toyoshima (part of the Molecular Psychiatry team at the RIKEN Brain Science Institute) uses iPS cells derived from patients with schizophrenia and developmental disorders to clarify disease mechanisms and potential therapeutic options. Dr. Toyoshima used our Guide-It Complete sgRNA Screening System to optimize their screen for high-efficiency guide RNAs (gRNAs).

Results

Five gRNAs were designed to target a gene of interest in human iPS cells and cleavage efficiency was confirmed in vitro using the Guide-It Complete sgRNA Screening System (Figure 1). Based on these results, two gRNAs (Nos. 2 and 4) were selected and used for knockout experiments in human iPS cells (Figure 2). Mutagenesis was confirmed with our Guide-It Mutation Detection Kit.

Figure 1. gRNA cutting efficiency revealed using the Guide-It Complete sgRNA Screening System. gRNAs No. 2 and 4 (highlighted in red boxes) demonstrated high cutting efficiency and were used in subsequent analyses.

Figure 2. Analysis of gRNA editing efficiency in human IPS cells.

Introduction

Dr. Kazuki Nakao (University of Tokyo) researches IVF and cryopreservation methods for transgenic mouse strains. He has kindly provided data from a recent experiment demonstrating the superiority of our Guide-it Recombinant Cas9 (Electroporation-Ready) relative to several competitors.

Results

Genomic editing of the tyrosinase locus was performed using electroporation of Guide-it Recombinant Cas9 (Electroporation-Ready) in C57BL/6N mouse embryos. Fetuses were sacrificed at 14.5 days and the number of albino fetuses was used as a marker for successful genomic modification.

Introduction

Dr. Yuichi Hiraoka and his colleagues [Laboratory of Genome Editing for Biomedical Research, Medical Research Institute, Tokyo Medical and Dental University (TMDU)] help breed and establish new strains of transgenic mice. They recently performed Cas9 microinjections in previously-frozen mouse embryos and have provided data demonstrating the high efficiency of our Guide-it Recombinant Cas9 (Electroporation-Ready).

Results

Genomic editing was performed to introduce knockin, knockout, SNP, or Flox sites using microinjection of a donor construct (Table II) and Guide-It Recombinant Cas9 (Electroporation-Ready) into C57BL/6 embryos. Successful editing and efficiency were determined by the proportion of pups that carried the targeted insert.

References

Aida, T. et al. Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biol. 16, 87 (2015).

Aida, T. et al. Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ. BMC Genomics 17, 979 (2016).