screens or custom cell line editing experiments is to determine if the target gene‐of‐interest (GOI) is expressed in the host cell and whether it is essential for cellular viability. Online tools are available and can be used (Chen et al. 2017) to help determine this. However, essentiality for some genes is context dependent (Wang et al. 2015) and as such caution on data interpretation is advised (Kimberland et al. 2018). Regardless, highly amplified genes in cancer cell lines appear to be unsuitable for gene KO studies, since simultaneous on‐target cleavage of a high number of alleles may affect cell viability. This has been shown to result in false positives/negatives in library screens where cell viability was used as an enrichment step (Aguirre et al. 2016; Munoz et al. 2016). One approach to address the challenge of essentiality for CRISPR screens is to use CRISPRi, which relies on dCas9 that does not alter the sequence of genomic DNA. However, CRISPRi may not be suitable for all targets, as it can downregulate multiple genes at bidirectional promoters (Rosenbluh et al. 2017).
To separate technical artifacts from true hits and to obtain a deeper understanding of how the target(s) of interest function, one can adopt a dual screening strategy, where two complementary screens (e.g. CRISPRi and CRISPRa) are performed in parallel (Jost et al. 2017; le Sage et al. 2020). Combining screening platforms substantially augments the quality and value of data derived from the screening campaigns, as well as providing novel insights not accessible when using one technology alone. For example, whereas CRISPRko will reveal targets where complete protein removal is required for a phenotype to emerge, CRISPRi could be used to investigate levels of repression needed to elicit a phenotypic response.
4.5.3 Controlling CRISPR Off‐Target Effects (OTEs) and Clonal Variations
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.
4.5.4 Deciding on Specific Quality Control Experiments on Engineered Cells
A key part of the research contract is to define quality control assays for the editing project, in addition to the prerequisite sequence confirmation of the editing site (on‐target editing). While these assays are not always essential, and will require additional effort and cost, certain assays can provide additional evidence supporting the quality of the resulting edited cells.
4.5.4.1 Confirmation of Gene KO at Protein Level
Ideally, gene KO clones with frameshift mutations should be further confirmed via protein analysis (e.g. Western) for depletion of the protein at the expected protein size, as long as a specific antibody is available. While this is observed for many genes in cellular KO experiments, deviations have been observed. In a recent study by Smits and colleagues, systematic characterization of frameshift KO mutations in HAP1 cell lines revealed that one‐third of the quantified targets were still detected at variable levels from low to original (Smits et al. 2019). Further studies of these frameshift KO clones, as well as other studies on genetically confirmed KO clones, revealed that residual levels of expression are either derived from introducing a premature stop codon, skipping of edited exon due to alternative mRNA splicing or from using an alternative initiation codon (Mou et al. 2017; Sharpe and Cooper 2017; Smits et al. 2019; Tuladhar et al. 2019). Dependent on the particular gene in study, and influenced by the location of the edited exon and the isoform expression profile in the cellular host, such truncated proteins may have full, partial, or even opposite activities (Smits et al. 2019; Tuladhar et al. 2019). Caution should be taken when interpreting KO effects of such clones. A practical approach to mitigate such effects is to analyze multiple independent clones derived from using gRNAs targeting different exons of the gene to obtain a concordant phenotypic observation. With a specific antibody, additional quality control assays can be carried out to analyze expression of the target protein in edited cells, including FACS analysis and cell staining. However, such assays only report on the presence/absence of the domain(s) the antibody recognizes, but not report on the size of the proteins/truncates detected, whereas Western analysis can report on both. Even with Western analysis, the results are impacted by epitope specificity of the antibody used. An N‐terminal‐specific antibody detects the full length and truncated proteins resulting from premature translation terminations; whereas a C‐terminal‐specific antibody enables detection of the full length and truncates resulted from in‐frame exon skipping as well as alternative translation initiations. In cases where a heterologous reporter gene (i.e. GFP, Luc) or an epitope tag is inserted in frame with an endogenous gene via knock‐in reactions, well‐validated reporter/epitope‐specific antibodies can be readily used in QC assays at protein levels.
4.5.4.2 Confirmation of Genetic Manipulation at RNA Level
Gene KO clones can also be QC analyzed at the mRNA level via RT‐PCR or RT‐qPCR. This is based on the assumption that mRNAs with frameshift indels are noncoding and are subjected to nonsense mediated decay (NMD) (Popp and Maquat 2016). While this is the case for many genes with frameshift indels in defined cells, some edited RNAs have been observed to escape NMD (Smits et al. 2019). Further analyses revealed that these NMD‐independent RNAs persist in cells via mechanisms involving alternative translation initiation, in‐frame exon skipping, or simply location of indel‐derived premature termination codon (Popp and Maquat 2016; Tuladhar et al. 2019). As such, RT‐qPCR is not commonly used as a quality control assay in gene KO experiments. Nevertheless, targeted RT‐PCR followed by gel electrophoresis or DNA sequencing analysis can reveal detailed information about variant RNA species transcribed from the edited gene, which can facilitate picking of appropriate cell clones for downstream functional analysis. It should be noted that RNA‐based QC assays may become essential when the edited site/sequence involves expression or function of a noncoding RNA (i.e. microRNA, lncRNA).
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