Many online tools are available for the design of gRNAs that achieve high on-target efficiency with relatively low off-target effects [1214]. We found that the use of more than one gRNA per gene locus can lead to high efficiency of deletion without increasing cell death. However, high editing efficiency depends primarily on the quality of the gRNA, and one single high-quality gRNA can also be as effective. The ideal experimental setup will therefore depend on the specific gene to be deleted and on the quality of the gRNAs. Furthermore, the gene structure should also be taken into account, for instance by avoiding exon-intron boundaries (which may lead to the excision of the intervening intron without significantly affecting protein expression), or alternative splice junctions. In this study, oligonucleotides specific for the gene of interest were designed using a gRNA design online tool (Dharmacon, IDT). These RNA oligonucleotides contain a 20 nt target-specific sequence with a protospacer and a 16 nt sequence complementary to an ATTO-550-labelled tracrRNA (Dharmacon, IDT). Sequences were then manually screened based on their on-target and off-target score (which should be ideally both >70) and their position in the gene body, to give preference to the first exons. Negative controls for this experimental design include mock transfected cells (electroporated without any reagent) or with Cas9 protein only, or transfected using a non-targeting scrambled gRNA or gRNAs against a gene different from the one under consideration and not associated with the phenotype to be observed.

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