CRISPR/Cas technology is a relatively simple and efficient method for targeted genome editing, directed by single-guide RNAs (sgRNAs). Designing an optimal sgRNA is the key to nonspecific endonuclease activity minimisation, which causes off-target effects. Due to complexity of sgRNA-DNA interactions algorithms are used to predict potential off-target sites in genome of a selected organism. To test, if mouse reference genome is an optimal choice for designing sgRNA in different mouse strains, Cas-OFFinder algorithm was used to predict off-target sites for different sgRNAs, using genomes of different mouse strains and the reference genome. We found that the reference genome is not always an optimal choice for sgRNA design and that off-target sites may differ between mouse strains and even between individuals of the same strain. Use of the reference genome is therefore suitable for sgRNA design in strains where specific genomes are unavailable, but more frequent off-target effects may be observed in such cases. Optimal choice for sgRNA design would be the genome of the targeted organism. In cases when individual genomes are not available, a genome of a closely related breed or strain to the selected organism should be used preferentially to the reference genome.
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