CRISPR Screening for Precision Cancer Medicine

Cancer is primarily a genetic disease, arising from mutations in the genetic code that aberrate cellular pathways governing cell growth and survival. Cancer treatment therefore aims to target the genes and their products that drive cancer progression. But despite recent advances in cancer therapeutics, a common issue is the variability in how a patient responds to a drug. For some patients, there is no clinical benefit, others may experience an adverse reaction to therapy, and a proportion may initially respond well to treatment but go on to develop drug resistance. Being able to predict how a patient will respond to a particular cancer drug or whether they will develop resistance allows for stratification of the patient cohort based on the drug response, helping clinicians identify the best treatment regimen.

This article discusses how CRISPR-Cas9 screening is being used to elucidate drug-gene interactions for clinical benefit, getting us closer to the holy grail of cancer treatment—personalized medicine and the rational design and application of therapies tailored to the genetics of individual tumors.

The complexity of cellular mechanisms governing cancer

The biological pathways that govern cancer progression and drug response, toxicity, and resistance are rarely controlled by a single gene, and interactions between genes do not always result in a predictable phenotype. Combining dysregulation of genes in multiple pathways can have unpredicted effects, which can be leveraged both by the cancer itself to drive growth, or to treat the disease. In the case of oncogenic growth, secondary mutations can lead to drug resistance by altering the efficacy of a drug, for example, a T790M mutation in EGFR results in an increased affinity for the receptor for ATP, meaning EGFR tyrosine kinase inhibitor drugs are outcompeted for binding.¹ Or genetic interactions can be leveraged to treat disease, such as synthetic lethality. In these cases, a single mutation alone is tolerated by the cell but can result in death if they occur in combination, such as genes functioning in DNA repair pathways. For example inhibition of PARP that functions in the base excision repair (BER) pathway is synthetic lethal with mutations in BRCA1/2, which are involved in double-strand break (DSB) repair.²

Knowledge of these complex drug-gene interactions has already led to the discovery of targeted therapies that are now available in the clinic—such as the PARP inhibitor olaparib, which is used to treat ovarian cancer patients with germline or somatic BRCA1/2 mutations.³ A deeper understanding of these interactions can enable future drug discovery and development, forming the basis of guidelines by which clinicians can tailor the treatment of their patients to the genetics of the disease being treated.

CRISPR-based screening for improving cancer treatment

Functional genomic screening with CRISPR-Cas9 offers a very promising route to this deeper understanding, in particular by allowing scientists to identify gene interactions that can sensitize patients to treatment with a drug, or by identifying potential candidate genes through which resistance mechanisms might occur. Pooled library screens using CRISPR-Cas9 are now increasingly popular. Requiring only basic molecular biology techniques and access to next-generation sequencing and bioinformatic analysis, they are a powerful tool for administering unbiased and systematic genome-wide loss of function studies. Using a library of sgRNA targeting some or all of the genes in the genome, the effect of gene modulation under screening conditions is observed, and hits from the screen recovered using massively parallel sequencing.

Delivery of a genome-wide sgRNA library by lentiviral vector targets the Cas9 endonuclease to introduce a double-strand break into the target gene resulting in complete knockout of the target gene. As well as gene knockout, the CRISPR system has been further developed with the use of a catalytically inactive version of Cas9 (dCas9) tethered to transcriptional repressors and activator domains that allow for inhibition and activation of gene expression with CRISPRi and CRISPRa. CRISPRi is reversible, inducible, and can achieve alternate levels of gene knockdown, thereby better mimicking the pharmacological effects of a drug.

Using CRISPR to identify novel drug targets and synthetic lethal interactions

The simplest CRISPR screen relies on proliferation or cell survival as the observed phenotype—any sgRNA that are depleted over the experiment indicate a gene that is required for cell survival. Screening conditions can involve treatment with a drug, or the presence of a known cancer driver mutation compared to wild-type control, and so this approach can be used to identify potential novel drug targets in otherwise intractable cancers. A good example of this is in the case of KRAS mutations, which for many years have defied the development of a small-molecule inhibitor. By identifying those genes, which if inhibited in combination with KRAS mutation leads to synthetic lethality, an alternative treatment could be identified.⁴ Similarly, knockdown of PI3Kd sensitizes acute lymphoblastic leukemia cells to dexamethasone, suggesting a drug synergy with the PI3Kd inhibitor CAL-101/idelalisib.⁵ CRISPR screening has been utilized to identify gene knockouts that sensitize tumors to treatment, such as Ptpn2, which significantly improved tumor response immunotherapy.⁶

CRISPR screening for potential drug-resistance mechanisms

By contrast to drug sensitization, sgRNAs that are enriched in survival assays can provide some insights into potential drug-resistance mechanisms. A genome-wide GeCKO library has been used to identify those genes that confer resistance to Quizartinib (AC220) in acute myeloid leukemia cells⁷ and use of a CRISPR library targeting 19,114 protein-coding genes found that knockout of specific zinc finger nucleases (ZFNs), post-translational modification enzymes, or microtubule components may modulate docetaxel resistance in castration-resistant prostate cancer cells.⁸ CRISPR screening has also been utilized to identify resistance mechanisms to the DNA topoisomerase inhibitor topotecan in ATM-deficient cells.⁹

Personalized medicine with CRISPR

In the short time since it’s development, the CRISPR technology has revolutionized functional genomics and high-throughput screening, providing a powerful tool allowing scientists to unpack the complex genetic relationships that govern cancer progression. CRISPR screens can not only aid in the identification of novel drug targets and synthetic lethal interactions, but also the genes that may aid in predicting patient responses to certain drugs, allowing patient stratification for optimal treatment regimens and clinical outcomes.

Key Takeaways

• CRISPR-based screening is helping scientists to identify the complex cellular relationships that govern cancer initiation and progression
• Identification of genes involved in drug resistance or sensitization can be used for optimizing treatment regimens and patient stratification
• The development of the CRISPR toolkit means that genome-wide screens can include gene knockout with wild-type Cas9, as well as modulation of gene expression with CRISPRi and CRISPRa
• CRISPRi may better mimic the pharmacological action of a drug due to the reversible and incomplete nature of knockdown compared to total knockout with CRISPR-ko

References

1. Yun, C. H. et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proceedings of the National Academy of Sciences of the United States of America 105, 2070–2075 (2008)

2. Pettitt, S. J. et al. Genome-wide and high-density CRISPR-Cas9 screens identify point mutations in PARP1 causing PARP inhibitor resistance. Nature Communications 9, (2018)

3. Moore, K. et al. Maintenance olaparib in patients with newly diagnosed advanced ovarian cancer. New England Journal of Medicine 379,2495–2505 (2018).

4. Aguirre, A. J. & Hahn, W. C. Synthetic lethal vulnerabilities in kras-mutant cancers. Cold Spring Harbor Perspectives in Medicine 8, (2018)

5. Kruth, K. A. et al. Suppression of B-cell development genes is key to glucocorticoid efficacy in treatment of acute lymphoblastic leukemia. Blood 129, 3000–3008 (2017)

6. Manguso, R. T. et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature 547, 413–418 (2017)

7. Hou, P. et al. A Genome-Wide CRISPR Screen Identifies Genes Critical for Resistance to FLT3 Inhibitor AC220. Cancer Research 77, 4402–4413 (2017)

8. Haldrup, J., Schmidt, L., Pedersen, J. S. & Sørensen, K. D. Abstract 5899: Genome-wide CRISPR-Cas9 screening identifies genetic vulnerabilities and potential therapeutic targets in castration resistant prostate cancer. in 5899–5899 (American Association for Cancer Research (AACR), 2018). 

9. Balmus, G. et al. ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks. bioRxiv 330043 (2018).