Fig. 2

Different workflows used in CRISPR screening and mutagenesis. The CRISPR screening procedures commence by selecting the appropriate screening system, offering various options: A CRISPRko, where Cas9 is employed to disrupt genes, resulting in the generation of premature stop codons or frameshift mutations; CRISPRa, involving the attachment of activation domains (e.g., VPR, VP64) to dCas9, resulting in enhanced transcription of target genes; CRISPRi, on the contrary, employs repression domains (e.g., KRAB) tethered to dCas9, leading to a reduction in the transcription of target genes; Base editing screen, which uses a base editor (e.g., cytosine deaminase or adenine deaminase) with or without a uracil DNA glycosylase inhibitor to induce mutations without causing double-strand breaks. Once the suitable CRISPR screening method is chosen, the gRNA library is introduced into cells, creating a genetically altered cell population. These cells are exposed to drugs to select for drug-resistant populations. Subsequently, the gRNAs are extracted from the cells, amplified via PCR, and their target genes are determined using next-generation sequencing. B On the other hand, CRISPR mutagenesis screening begins with a gRNA library designed to induce in-frame mutations in the target protein coding sequence. After transducing the cells with the gRNA library, viable cells with protein variants are subjected to drug treatment, both with and without the drug. Activity-based cell sorting is used to enrich cells carrying mutations that make the drug ineffective, thereby identifying drug-resistant cells. Finally, the enriched cells are genotyped using deep sequencing to analyze structural changes and detect any escape mutants. Reprinted from [14] with permission from Cell Press