Applications of the CRISPR-Cas9 system in cancer biology

Abstract

The prokaryotic type II CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system is rapidly revolutionizing the field of genetic engineering, allowing researchers to alter the genomes of a large range of organisms with relative ease. Experimental approaches based on this versatile technology have the potential to transform the field of cancer genetics. Here, we review current approaches for functional studies of cancer genes that are based on CRISPR-Cas, with emphasis on their applicability for the development of next-generation models of human cancer.

Figure 1. Genome engineering utilizing the CRISPR-Cas9 system

a | DNA double-strand breaks (DSBs) can be repaired by two cellular DNA repair pathways: the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway. Repair via the NHEJ pathway, which is error-prone, frequently leads to insertion or deletion mutations (indels) that can lead to disrupting frameshift mutations and the generation of premature stop codons. Alternatively, in the presence of an exogenous donor DNA template, the DSB can be repaired via the HDR pathway, which can be utilized for engineering precise DNA modifications. b | The S. pyogenes-derived Cas9 RNA-guided DNA endonuclease is localized to a specific DNA sequence via a single guide RNA (sgRNA) sequence, which base-pairs with a specific target sequence that is adjacent to a protospacer adjacent motif (PAM) sequence in the form of NGG or NAG. Cas9-mediated induction of a DSB in the DNA target sequence leads to indel mutations via NHEJ or precise gene modification via HDR.

Figure 1. Genome engineering utilizing the CRISPR-Cas9 system

a | DNA double-strand breaks (DSBs) can be repaired by two cellular DNA repair pathways: the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway. Repair via the NHEJ pathway, which is error-prone, frequently leads to insertion or deletion mutations (indels) that can lead to disrupting frameshift mutations and the generation of premature stop codons. Alternatively, in the presence of an exogenous donor DNA template, the DSB can be repaired via the HDR pathway, which can be utilized for engineering precise DNA modifications. b | The S. pyogenes-derived Cas9 RNA-guided DNA endonuclease is localized to a specific DNA sequence via a single guide RNA (sgRNA) sequence, which base-pairs with a specific target sequence that is adjacent to a protospacer adjacent motif (PAM) sequence in the form of NGG or NAG. Cas9-mediated induction of a DSB in the DNA target sequence leads to indel mutations via NHEJ or precise gene modification via HDR.

Figure 2. Applications of the CRISPR-Cas9 system in cancer biology

a | CRISPR-mediated genome engineering of embryonic stem (ES) cells or genetically engineered mouse model (GEMM)-derived ES cells can be utilized for rapidly generating novel GEMMs or non-germline GEMMs (nGEMMs) of cancer harbouring multiple genetic alterations, such as constitutive or conditional knockout and knock-in alleles, endogenous synthetic tags or reporters, non-coding single nucleotide polymorphisms (SNPs) and genomic rearrangements, as well as a combination of all of these via re-engineering of ES cells or multiplex CRISPR-mediated genome engineering. b | CRISPR-mediated somatic genome engineering in vivo can be utilized to rapidly generate cohorts of tumor-bearing mice for studying both basic and translational aspects of cancer biology. For example, the CRISPR-Cas9 system can be deployed in vivo for generating cohorts of personalized mice based on the exact complement of mutations observed in individual patients. c | The CRISPR-Cas9 system can serve as an important conduit between the bench and the bedside. The combination of sophisticated molecular profiling technologies with CRISPR-based genome engineering technologies will allow researchers to generate personalized experimental platforms that can be utilized for rapidly and systematically identifying novel genotype-specific vulnerabilities through a battery of cell-based and in vivo assays.

Figure 2. Applications of the CRISPR-Cas9 system in cancer biology

a | CRISPR-mediated genome engineering of embryonic stem (ES) cells or genetically engineered mouse model (GEMM)-derived ES cells can be utilized for rapidly generating novel GEMMs or non-germline GEMMs (nGEMMs) of cancer harbouring multiple genetic alterations, such as constitutive or conditional knockout and knock-in alleles, endogenous synthetic tags or reporters, non-coding single nucleotide polymorphisms (SNPs) and genomic rearrangements, as well as a combination of all of these via re-engineering of ES cells or multiplex CRISPR-mediated genome engineering. b | CRISPR-mediated somatic genome engineering in vivo can be utilized to rapidly generate cohorts of tumor-bearing mice for studying both basic and translational aspects of cancer biology. For example, the CRISPR-Cas9 system can be deployed in vivo for generating cohorts of personalized mice based on the exact complement of mutations observed in individual patients. c | The CRISPR-Cas9 system can serve as an important conduit between the bench and the bedside. The combination of sophisticated molecular profiling technologies with CRISPR-based genome engineering technologies will allow researchers to generate personalized experimental platforms that can be utilized for rapidly and systematically identifying novel genotype-specific vulnerabilities through a battery of cell-based and in vivo assays.

Figure 2. Applications of the CRISPR-Cas9 system in cancer biology

a | CRISPR-mediated genome engineering of embryonic stem (ES) cells or genetically engineered mouse model (GEMM)-derived ES cells can be utilized for rapidly generating novel GEMMs or non-germline GEMMs (nGEMMs) of cancer harbouring multiple genetic alterations, such as constitutive or conditional knockout and knock-in alleles, endogenous synthetic tags or reporters, non-coding single nucleotide polymorphisms (SNPs) and genomic rearrangements, as well as a combination of all of these via re-engineering of ES cells or multiplex CRISPR-mediated genome engineering. b | CRISPR-mediated somatic genome engineering in vivo can be utilized to rapidly generate cohorts of tumor-bearing mice for studying both basic and translational aspects of cancer biology. For example, the CRISPR-Cas9 system can be deployed in vivo for generating cohorts of personalized mice based on the exact complement of mutations observed in individual patients. c | The CRISPR-Cas9 system can serve as an important conduit between the bench and the bedside. The combination of sophisticated molecular profiling technologies with CRISPR-based genome engineering technologies will allow researchers to generate personalized experimental platforms that can be utilized for rapidly and systematically identifying novel genotype-specific vulnerabilities through a battery of cell-based and in vivo assays.

Figure 2. Applications of the CRISPR-Cas9 system in cancer biology

a | CRISPR-mediated genome engineering of embryonic stem (ES) cells or genetically engineered mouse model (GEMM)-derived ES cells can be utilized for rapidly generating novel GEMMs or non-germline GEMMs (nGEMMs) of cancer harbouring multiple genetic alterations, such as constitutive or conditional knockout and knock-in alleles, endogenous synthetic tags or reporters, non-coding single nucleotide polymorphisms (SNPs) and genomic rearrangements, as well as a combination of all of these via re-engineering of ES cells or multiplex CRISPR-mediated genome engineering. b | CRISPR-mediated somatic genome engineering in vivo can be utilized to rapidly generate cohorts of tumor-bearing mice for studying both basic and translational aspects of cancer biology. For example, the CRISPR-Cas9 system can be deployed in vivo for generating cohorts of personalized mice based on the exact complement of mutations observed in individual patients. c | The CRISPR-Cas9 system can serve as an important conduit between the bench and the bedside. The combination of sophisticated molecular profiling technologies with CRISPR-based genome engineering technologies will allow researchers to generate personalized experimental platforms that can be utilized for rapidly and systematically identifying novel genotype-specific vulnerabilities through a battery of cell-based and in vivo assays.

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