Читать книгу Genome Editing in Drug Discovery - Группа авторов - Страница 68

Despite the remarkable specificity of the first and commonly used CRISPR/Cas9 system derived from Streptococcus pyogenes (SpCas9), varying levels of OTEs have been observed (Cho et al. 2014; Fu et al. 2013; Hsu et al. 2013; Lin et al. 2014; Pattanayak et al. 2013). These effects can be exacerbated when combined with additional sources of experimental variation, such as clonal variation in the cellular system. In cases where gene KO efficiency is high (e.g. >90%), bulk cell populations can be readily assayed without further enrichment; this is analogous to experimental approaches applied to siRNA/shRNA gene knockdown. Certain CROs offer gene KO in bulk for cell lines and ship the KO cell populations as frozen stocks (e.g. Synthego). However, when the editing efficiencies are lower and bulk assays are not feasible, especially if using iPSC or cell lines, cellular cloning will be needed. Successful on‐target changes in genome‐edited cells are typically monitored by targeted analysis of the genomic loci by a number of assays routinely offered by CROs, including the T7 endonuclease‐based Surveyor assay (Qiu et al. 2004), or the sequencing‐based TIDE assay (Brinkman et al. 2014). A comprehensive assessment of OTEs would require a genome‐wide approach (Zischewski et al. 2017), which are typically resource intensive. Even mapped, it is still unclear to what extent the detected OTEs will impact interpretation of the experimental results. More practically, OTEs can be controlled by analyzing multiple independent clones that are derived either from using the same gRNAs (or a mix of 2–3 gRNAs) or ideally from using different gRNAs. Given that each gRNA has its unique off‐target sites to which the OTEs are most likely to occur, different gRNAs targeting the same gene or locus thus will most likely have different OTE profiles. Further, it is a good practice to also obtain “wild type” (WT) clones for use as controls. These WT clones are derived from the same CRISPR editing reaction yet are wild type at the targeting site, but may carry indels at off‐target sites. Such WT clones thus offer better controls than unedited cells since they have the potential to account for OTEs. The number of independent clones to analyze will vary dependent on a balance of quality, time, and cost. Scientifically, it depends on multiple factors including homogeneity of the parental cells, since clonal heterogeneity can lead to confounding or even invalid results (Ben‐David et al. 2018). Also, editing specificity and efficiency need to be considered. For practicality, it is commonplace to analyze 3–5 independent edited clones along with 3–5 WT clones. Statistically, the more clones analyzed, the higher confidence one will have in the resulting data.