Fig. 4.

Schematic diagram of in vitro or in vivo CRISPR screening. a CRISPR screening begins by synthesizing oligonucleotide pools containing single guide RNA sequences and cloning them into lentiviral vectors. Lentiviruses then infect cells expressing Cas9 at low multiplicity of infection. After selection, the pool contains cells with different gene knockouts, which can be subsequently used in various screening methods. b In vitro screening is performed by culturing tumor cells under selective pressure such as drug treatment. c In vivo screening transplants the transfected cell population into immunodeficient mice in situ or subcutaneously. d Patient-derived xenotransplantation (PDX) is achieved by transplanting the patient’s tumor into immunodeficient mice. The PDX tumor is harvested, cultured in vitro, and genetically modified to evaluate tumor growth and response to treatment