What should I consider when planning a CRISPR screen?

There are several factors one should consider when planning a CRISPR screen. One factor is ensuring the relevance and interpretability of the results. It's crucial to carefully select and optimize the screening model. Using multiple variants of the same model (like cell lines with different genetic backgrounds) can improve the understanding of the findings. Another factor to consider are biological replicates, as the same genetic change may result in different phenotypic outcomes depending on the genetic makeup. CRISPR screens should ideally include at least three biological replicates. Another factor is choosing the Cas9 nuclease type depending on whether a DNA-free (mRNA, protein) or vector-based (e.g., lentivirus) approach is preferred. One should also consider the amount of the detection assay required after editing. For screening, visualizing a phenotype can indicate the desired outcome. However, to confirm the success of the CRISPR-Cas9 reaction in introducing deletions and insertions into the cell population, DNA mismatch detection assays like Surveyor are commonly used, followed by Sanger Sequencing and western blot.