Cancer CRISPR Screens In Vivo

Abstract

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.

Keywords: CRISPR screen; cancer; functional genomics; in vivo.

Figure 1. Functional cancer genomics with in vivo CRISPR screening

In vivo CRISPR screening is a powerful, flexible tool to dissect important processes in cancer. Genome-wide CRISPR screens have illuminated novel regulators of metastasis, malignant transformation, immune evasion, and drug resistance. Moving forward, in vivo CRISPR screens may improve our understanding of other processes, such as angiogenesis, genome instability, and metabolic programming.

Figure 2 Key Figure. Three modes of CRISPR screening

A. To perform an in vitro CRISPR screen, the desired sgRNA library must first be cloned into expression vectors. Lentiviral vectors are commonly used, as they can stably integrate into the host genome. After a selection phase to enrich for a desired phenotype, the sgRNA cassettes are amplified from genomic DNA and sequenced to identify the top candidate genes. B. Indirect in vivo screens follow the same steps asin vitro studies, but the selection phase occurs within a recipient animal. Following transplantation of the mutagenized cell pool into mice, different mutants will become enriched. In the case of a tumorigenesis screen, highly abundant sgRNAs within the resultant tumors would be brought forward as candidate tumor suppressors. C. For direct in vivo screens, CRISPR mutagenesis occurs at the autochthonous target organ site instead of in culture. Lentiviral and adeno-associated viral (AAV) approaches have both been successfully used for multiplexed direct in vivo mutagenesis. Intravenous, intracranial, and intratracheal viral injections can drive tumorigenesis from the liver, brain, and lung, respectively. Since AAVs do not integrate into the genome, capture sequencing must be performed to readout the results of the screen (i.e. highly abundant indel variants).

Figure 3. Applications and extensions of direct in vivo CRISPR screens

A. Direct in vivo CRISPR screens can be readily used to generate personalized mouse avatars. CRISPR libraries can be customized to the mutations present within a given patient’s tumor. Following autochthonous mutagenesis, therapeutic candidates can be evaluated using the mouse avatars, informing clinical decision-making. B. Higher dimensional screens (i.e. double, triple, n-tuple knockouts) using CRISPR-Cpf1 offer an elegant high-throughput approach to investigate genetic interactions. Such studies could uncover synergistic driver mutations and synthetically lethal combinations, which may help to inform patient prognostication and to identify novel therapeutic vulnerabilities. C. Direct in vivo CRISPR mutagenesis drives the formation of genetically complex multi-clonal tumors. Coupled with single-cell capture sequencing and RNA sequencing, direct in vivo CRISPR screens offer a powerful approach for the generation and subsequent dissection of tumor heterogeneity. D. In vivo CRISPR screens thus far have lacked spatial resolution, as sgRNA sequencing and/or capture sequencing is performed on genomic DNA extracted from dissociated cell suspensions. By combining direct in vivo CRISPR mutagenesis with multiplexed in situ hybridization, it may be feasible to perform clonal analysis and phenotype-genotype matching on tissue sections.