CRISPR/Cas9 is revolutionary for gene editing and gene function assays, and our Guide-it technology powers these experiments in laboratories around the world. Several customers have kindly provided data they've generated with our products, so look below to see why scientists use Guide-it products.
Optimizing a screen for high-efficiency guide RNAs
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.
Electroporation of recombinant Cas9 in mouse embryos
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.
Manufacturer
Cas9 concentration (ng/µl)
Number of embryos subjected to electroporation
Number of implanted electroporated embryos
Number of fetuses
Number of albino fetuses
Takara Bio
25
60
60
12
9
100
60
60
7
7
Company A
25
60
60
13
2
100
60
60
23
12
400
40
40
7
4
Company B
25
60
59
22
0
Table I. Results of tyrosinase genome editing using Takara Bio's Guide-it Recombinant Cas9 (Electroporation-Ready) versus two competitors' products. Takara Bio's recombinant Cas9 resulted in successful genome editing in 75% and 100% of embryos (with 25 and 100 ng/µl Cas9, respectively), compared to a success rate of 15%, 52%, and 57% using Company A's product (with 25, 100, and 400 ng/µl Cas9, respectively). Notably, Company B had a 0% success rate (with 25 ng/µl Cas9).
Microinjection of recombinant Cas9 in mouse embryos
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.
Gene name
Type (insertion cassette/deletion length)
Donor
Litter size
Pups containing modification
Efficiency
Gene A
Knockin (insert length: 3 kb)
Plasmid
9
4
44%
Gene B
Knockin (insert length: 7.5 kb)
Plasmid
11
4
36%
Gene C
Knockout (deletion length: 51 kb)
Oligo DNA donor
14
4
28%
Gene D
SNP
Oligo DNA donor
8
2
25%
Gene E
Flox
Plasmid
6
2
33%
Gene F
Flox
Plasmid
8
2
25%
Table II. Results of several types of genome editing performed using microinjections of our Guide-It Recombinant Cas9 (Electroporation-Ready) into C57BL/6 fertilized embryos. Takara Bio's recombinant Cas9 demonstrated comparatively high editing efficiencies across all four types of genomic editing performed.
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 Genomics17, 979 (2016).
