CRISPR design and synthesis 3-4 weeks
For each target two or three different guide RNAs (sgRNA) sequences will be identified.
- Design proposal for 2-3 custom sgRNAs, targeting the gene or region of interest
- Analysis of potential off-targets
- Synthesis of sgRNA
- For knock-in models: design and synthesis of template DNA; either a large oligo nucleotide or small targeting vector containing the desired knock-in
- Co-injection of sgRNAs, Cas9 protein and template DNA in C57Bl/6 zygotes
- Transfer of injected zygotes into foster mice
- Genotyping and Sequencing of offspring to identify correctly modified target regions
Depending on the specificity of the selected sgRNA sequences, a thorough analysis of off-target effects is recommended. Please inquire for more details.