Cancer CRISPR Screens In Vivo

In vivo CRISPR screens enable high-throughput interrogation of complex processes in cancer. Direct autochthonous models recapitulate human cancer by maintaining the native microenvironment. Direct in vivo CRISPR technologies can empower patient-specific cancer modeling for precision medicine. Clustered regularly interspaced short palindromic repeats (CRISPR) screening is a powerful toolset for investigating diverse biological processes. Most CRISPR screens to date have been performed with in vitro cultures or cellular transplant models. To interrogate cancer in animal models that more closely recapitulate the human disease, autochthonous direct in vivo CRISPR screens have recently been developed that can identify causative drivers in the native tissue microenvironment. By empowering multiplexed mutagenesis in fully immunocompetent animals, direct in vivo CRISPR screens enable the rapid generation of patient-specific avatars that can guide precision medicine. This Opinion article discusses the current status of in vivo CRISPR screens in cancer and offers perspectives on future applications. Clustered regularly interspaced short palindromic repeats (CRISPR) screening is a powerful toolset for investigating diverse biological processes. Most CRISPR screens to date have been performed with in vitro cultures or cellular transplant models. To interrogate cancer in animal models that more closely recapitulate the human disease, autochthonous direct in vivo CRISPR screens have recently been developed that can identify causative drivers in the native tissue microenvironment. By empowering multiplexed mutagenesis in fully immunocompetent animals, direct in vivo CRISPR screens enable the rapid generation of patient-specific avatars that can guide precision medicine. This Opinion article discusses the current status of in vivo CRISPR screens in cancer and offers perspectives on future applications. an endonuclease that is directed to specific sites in the genome by CRISPR spacers, where it induces double-stranded breaks in the target DNA. segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of ‘spacer DNA’ from previous exposures to a bacteriophage virus or plasmid. The CRISPR–Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present in plasmids and phages. Cas proteins use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms. By delivering the Cas9 nuclease and appropriate guide RNAs (gRNAs) into a cell, the cell’s genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. also known as Cas12a; a CRISPR-guided endonuclease that can be utilized for targeted genome editing in diverse species. Unlike Cas9, Cpf1 does not require a tracrRNA for DNA cleavage, and has the capability to autonomously process crRNA arrays. the short RNA sequence that directly guides CRISPR nucleases to target sites in the genome that is independent of the scaffold sequence present in full-length sgRNAs. catalytically dead Cas9 nuclease that cannot generate double-stranded breaks in DNA; commonly tethered to other proteins to enable programmable targeting of the tethered enzymes. a CRISPR screen in which two genes are simultaneously interrogated rather than a single gene. a mutation that is causally implicated in oncogenesis. It confers a growth advantage on the cancer cell and is favored in the microenvironment of the tissue in which the cancer arises. also known as genome editing with engineered nucleases (GEEN); a type of genetic engineering in which DNA is inserted, deleted, or replaced in the genome of an organism using engineered nucleases, or ‘molecular scissors’. a short synthetic RNA comprising a ‘scaffold’ sequence necessary for Cas9 binding and a user-defined ∼20-nucleotide ‘spacer’ or ‘targeting’ sequence that defines the genomic target to be modified. a CRISPR screen in which three or more genes are simultaneously interrogated, enabling the investigation of complex multigenic phenotypes. by generating libraries of sgRNAs targeting different genes, a CRISPR screen can be performed to assess the importance of these genes towards a given phenotype. With in vivo CRISPR screens, the selection phase occurs inside a living organism; for instance, a mouse. a gene that has the potential to cause cancer. In tumor cells, oncogenes are often mutated or expressed at high levels. a gene that researchers attach to a regulatory sequence to facilitate readout of gene regulatory activity. Common examples include GFP and firefly luciferase. a term generally interchangeable with gRNA in genome editing with the CRISPR–Cas9 system. arises when a combination of mutations in two or more genes leads to cell death, whereas a mutation in only one of these genes does not. In a synthetic lethal genetic screen, it is necessary to begin with a mutation that does not kill the cell, although it may confer a phenotype (e.g., slow growth) and then systematically test other mutations at additional loci to determine which ones confer lethality. Synthetic lethality indicates functional relationships between genes. refers to the interaction between the host immune system and a tumor. In different contexts, the immune system can have opposing roles towards cancer progression. Cancer immunotherapies aim to tip the scales towards immune-mediated elimination of the tumor. the stroma and supporting milieu surrounding the tumor, usually comprising fibroblasts, immune cells, and endothelial cells. also known as an antioncogene; a gene that protects a cell from one step on the path to cancer. Mutations that inactivate tumor suppressors will contribute to cancer progression.