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CRISPR/Cas9: From Genome Engineering to Drug Discovery and Therapy

Functionalizing the cancer genome is crucial for identifying appropriate oncogene and non-oncogene targets which could afford therapeutic benefit in cancer patients. The oncogenic activity of driver mutations can be modeled by gene targeting using Cre and Flp recombinases as well as gene editing through zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Recently, a new and powerful genome editing technology based on the bacterial CRISPR endonuclease system was discovered. CRISPR is a versatile platform that enables us to activate, knockout, and introduce precise mutations in genes. Its deployment in cancer research as the genome editing tool and screening tool will dramatically accelerate the pace of cancer target discovery and target validation.

CRISPR-Cas9 for genome engineering

The RNA-guided endonuclease Cas9 (CRISPR-associated protein 9), a component of the type II CRISPR (clustered regularly interspaced short palindromic repeats) system of bacterial host defense, is a powerful tool for genome editing. Ectopic expression of the Cas9 and a single guide RNA (sgRNA) is sufficient to direct the formation of a DNA double-strand break (DSB) at a specific region of interest. In the absence of a homology-directed repair DNA template, these DSBs are repaired in an error-prone manner by means of the nonhomologous end-joining pathway to generate an assortment of short deletion and insertion mutations in the vicinity of the sgRNA recognition site. This method has been widely used to generate gene-specific knockouts in multiple biological systems.

CRISPR/Cas9: From Genome Engineering to Drug Discovery and Therapy Figure 1. CRISPR/Cas9 Tools for Genome Engineering.

One major application of CRISPR/Cas9 in mammalian cells is the regulation of gene expression. CRISPR/Cas9 is highly efficient for gene knockout in mammalian cells when Cas9 is targeted to the exon regions of a gene. In this condition, cutting by Cas9 and subsequent repair by NHEJ leads to indel mutations that often cause frame shift in the target gene. This results in either the production of an on functional, truncated protein or the degradation of mutant mRNA through nonsense-mediated mRNA decay. While RNAi rarely achieves complete silencing, CRISPR/Cas9 can generate true nulls.

Recently, studies have shown the use of CRISPR-Cas9 mutagenesis for genetic screens in mammalian cell culture, which have relied on sgRNA libraries which target constitutive 5’ coding exons to achieve gene inactivation. The capabilities of CRISPR-based genetic screens are particularly evident in the setting of positive selection, such as identifying mutations that confer drug resistance. In negative selection screens, it has been demonstrated that sgRNA hits are statistically enriched for essential gene classes (ribosomal, DNA replication factors and RNA processing).

CRISPR/Cas9 as a tool for drug discovery

The recent development of easily programmable RNA-guided nucleases, which are derived from microbial adaptive immune systems, has revolutionized the molecular toolbox for mammalian genome engineering. The CRISPR/Cas9 systems stand poised to transform a number of stages of drug discovery and development by enabling fast and accurate alterations of genomic information in mammalian model systems and human tissues. Furthermore, direct somatic editing in patients will, eventually, radically change the druggable space by enabling the targeting of nearly any entity, including the introduction of corrective mutations and the modification of regulatory elements or splicing patterns. Following the description of a two-component sgRNA–Cas9 complex to introduce DSBs in an RNA-guided manner, many studies have shown ingenious applications and uncovered orthogonal immune systems, together enabling nearly unlimited genome engineering opportunities (Figure 2).

CRISPR/Cas9: From Genome Engineering to Drug Discovery and Therapy Figure 2. The pipeline of CRISPR–Cas-assisted drug discovery.

Gene knockout by CRISPR–Cas has proved to be efficacious in virtually all cell types, including induced pluripotent stem cells (iPSCs), primary immune cells and cancer-specific organoids. Therefore, knockout based target discovery efforts are no longer limited to specialized cell lines, such as the haploid lines that were previously used for gene trap experiments, and can instead be performed in the cell type that is most appropriate for the disease of interest. For instance, if a panel of tumor-derived lines is thought to be sensitized to a drug candidate through a genetic lesion, CRISPR–Cas-mediated gene knockout can directly test the hypothesis of synthetic lethality. Such isogenic knockouts allow researchers to rapidly establish causative roles for oncogenes, tumor suppressors and other factors in a defined context, thereby removing secondary differences.

Large-scale functional screening with CRISPR–Cas is simultaneously expanding and evolving, as researchers find the advantages and disadvantages of different screening systems. Until recently, systematic loss‑of‑function studies focused on insertional mutagenesis screens or genome-wide RNAi screens in haploid human cell lines. CRISPR–Cas screens have rapidly been adopted in various contexts due to the simplicity of designing potent sgRNAs and the ability to apply the system to nearly any cell type or tissue. Large-scale screens typically depend on pooled lentiviral libraries of sgRNAs, often achieving robust hit identification by including 3–10 sgRNAs per gene. The procedure of CRISPR–Cas-based screens is very similar to that of short hairpin RNA (shRNA) screens. A pool of cells that co-express Cas9 and the sgRNA library is subjected to the desired phenotypic selection, and high-throughput DNA sequencing of the sgRNA cassette is used to identify sgRNAs that were enriched or depleted during the treatment.

Using CRISPR/Cas9 to make therapeutics

Although Cas9 has been widely used as a research tool, a particularly exciting future direction is the development of Cas9 as a therapeutic technology for treating genetic disorders. For a monogenic recessive disorder due to loss-of-function mutations (such as sickle-cell anemia, cystic fibrosis, or Duchenne muscular dystrophy), Cas9 may be used to correct the causative mutation. This has a number of advantages over traditional methods of gene augmentation that deliver functional genetic copies through viral vector-mediated overexpression—particularly that the newly functional gene is expressed in its natural context. For dominant-negative disorders in which the affected gene is haplosufficient, it is also possible to use NHEJ to inactivate the mutated allele to achieve therapeutic benefit.

In addition to repairing mutations underlying inherited disorders, Cas9-mediated genome editing may be used to introduce protective mutations in somatic tissues to combat nongenetic or complex diseases. For instance, NHEJ-mediated inactivation of the CCR5 receptor in lymphocytes could be a viable strategy for circumventing HIV infection, while deletion of PCSK9 or angiopoietin could provide therapeutic effects against statin-resistant hyperlipidemia or hypercholesterolemia. Cas9 could be used beyond the direct genome modification of somatic tissue, such as for engineering therapeutic cells. Chimeric antigen receptor (CAR) T cells can be modified ex vivo and reinfused into a patient to specifically target certain cancers. The ease of design and testing of Cas9 may also facilitate the treatment of highly rare genetic variants via personalized medicine. Supporting these tremendous possibilities are many animal model studies and clinical trials using programmable nucleases that provide important insights into the future development of Cas9-based therapeutics.


  1. Fellmann C, et al. Cornerstones of CRISPR-Cas in drug discovery and therapy. Nature Reviews Drug Discovery, 2017, 16(2):89-100.
  2. Luo J. CRISPR/Cas9: From Genome Engineering to Cancer Drug Discovery. Trends in Cancer, 2016, 2(6):313-324.
  3. Hsu P D, et al. Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell, 2014, 157(6):1262-1278.
  4. Shi J, et al. Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains. Nature Biotechnology, 2015, 33(6):661-667.
For research use only. Not intended for any clinical use.

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