CRISPR/Cas9 for the Clinician: Current uses of gene editing and applications for new therapeutics in oncology


Julia Boland MD13; Elena Nedelcu MD2

Perm J 2020;24:20.040 [Full Citation]
E-pub: 12/09/2020


Precise genomic editing has given rise to treatments in previously untreatable genetic diseases and has led to revolutions in treatment for cancer. In the past decade, the discovery and development of clustered regularly interspaced short palindromic repeats (CRISPR) technologies has led to advances across medicine and biotechnology. Specifically, the CRISPR/Cas9 system has improved translational discovery and therapeutics for oncology across tumor types. In this review, we briefly summarize the history and development of CRISPR, explain CRISPR-Cas systems and CRISPR gene editing tools, highlight the development and application of CRISPR technologies for translational and therapeutic purposes in different oncologic tumors, and review novel treatment paradigms using CRISPR in immuno-oncology, including checkpoint inhibitors and chimeric antigen receptor T cell therapy.


In 2020, the US is expected to have over 600,000 deaths due to cancer.1 To date, treatment has largely focused on surgery, chemotherapy, and targeted therapies. Genetic mutations are among the most common causes of cancer. Therefore, gene therapy has a potential to guide therapy in oncologic patients in the future. Recently, the gene-editing tool CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has been used in oncology translational research, and therapeutics. CRISPR is derived from the natural adaptive immune system of bacteria. Current applications of research in CRISPR have been in studying gene knockout in cancers, editing mutated genes, and engineering T cells for chimeric antigen receptor T cell (CAR-T) therapy. This review summarizes CRISPR techniques and highlights the applications of the technology to cancer therapeutics.


The family of repetitive, mobile DNA sequences in prokaryotes was first described in 1987 in Escherichia coli and later named CRISPR.2,3 Some bacteria have CRISPR present, and other species within the same family do not contain CRISPR; additionally, unrelated species have been found to harbor identical CRISPR sequences.2 CRISPR sequences are a naturally occurring phenomenon and are protective against bacteriophages and conjugative plasmids.4 In microbiology, CRISPR sequences demonstrate a record of past infections and can be used to fight those pathogens in the future by direct degradation of foreign genome.4 To date, an array of invertebrates and vertebrates, bacteria, and plants have had their genomes edited by CRISPR.5

The Streptococcus pyogenes CRISPR system consists of precursor-CRISPR RNA, which is cleaved to form single-guide RNA (sgRNA), which is the mature CRISPR RNA.6 sgRNA contains complementary DNA to the target site and hybridizes with trans-activating CRISPR RNA, which then forms a complex with CRISPR-associated protein 9 (Cas9).6 The CRISPR-Cas9-sgRNA complex then edits the genome of interest. There are many Cas proteins; however, Cas9 is the most efficient and widely used.7 The Cas9 protein acts as scissors to cleave the targeted DNA sequence.7 Cas9 cuts 3 to 4 nucleotides upstream from the protospacer-adjacent motif.7

Genetics, lifestyle, and environmental components contribute to cancer risk through carcinogenesis and chromosomal damage. One manifestation of this is via double-strand DNA breaks (DSBs), which are common occurrences in cells and which play a key role in cancer development. To potentially counter this, repair mechanisms may be used to influence the applicability of CRISPR. Two main methods of repair are seen in eukaryotic cells: nonhomologous end joining (NHEJ) and homology-directed repair (HDR), most commonly as homologous recombination (Figure 1). When cells use each mechanism of repair is still debated; however, if a cell is in S phase, where the sister chromatid is nearby to act as a donor template, HDR is the preferred mechanism of repair.8 NHEJ is error prone and can lead to insertion-deletion or frameshift mutations; it is therefore the less preferred mechanism for CRISPR gene editing.5 However, NHEJ is efficiently used with CRISPR for gene knockout studies. When HDR is preferred in CRISPR, such as in gene editing for genetic diseases, it is possible to use an antagonist of an enzyme required for the NHEJ pathway because the two mechanisms naturally compete with each other to repair the DSBs.9


Figure 1. Pathway for dsDNA break in CRISPR. NHEJ = Non-homologous end joining; HDR = Homology-directed repair.

There are various methods of delivery and carriers of the CRISPR-Cas9 system. The delivery of sgRNA and Cas9 can be achieved via viral vectors, plasmid microinjection or lipofection of the sgRNA-Cas9 complex, and cell-penetrating peptides.10 Viral vectors, including the adeno-associated virus and lentivirus, allow for introduction of exogenous DNA and incorporation via HDR.11 The delivery mechanisms vary on efficiency and gene editing errors.

ReviewTranslational CRISPR Research in Oncology

Lung adenocarcinoma (ADC) is the leading cause of cancer death among men and women in the US.1 Lung cancer is broadly divided into small cell lung cancer and non-small cell lung cancer. Non-small cell lung cancer compromises the majority (80%) of cases and consists of histological types: large cell carcinoma, squamous cell carcinoma, and adenocarcinoma. Lung adenocarcinoma is the most common type diagnosed in both men and women.12 KRAS and EGFR are major genetic targets in lung adenocarcinoma. Patients with EGFR mutations can be susceptible to targeted therapy with gefitinib and erlotinib, which are first-generation competitive, reversible inhibitors of the EGFR-tyrosine kinase. These therapies have provided significant improvement in the progression-free survival of EGFR-mutated lung adenocarcinoma; however, these agents have not shown significant benefit to overall survival.13 Moreover, many of these patients eventually develop resistance to these first-line agents.13 The most common mechanism of EGFR tyrosine kinase inhibitor (TKI) resistance is via the T790M mutation in exon 20 of the EGFR gene, followed by MET amplification.14 Approximately 60% of patients develop T790M mutations while on TKI therapy.15 In a recent study of the third-generation EGFR TKI, osimertinib, CRISPR/Cas9 was used for gene knockout of MEK/ERK signaling in lung adenocarcinoma resistant to osimertinib.16 Viral production and transduction methods were used for this study.16

In KRAS-mutant lung ADC, the overall response rate to treatments remains low despite the introduction of novel agents, including immunotherapies.17 Enhancing the immune responsiveness to checkpoint inhibitor therapy with epigenetic modification is one method of improving response to therapy. Epigenetic modifications consist of DNA base methylation and histone modification, which affects gene expression.18 A study of KRAS-mutant lung ADC constructed an in vivo CRISPR screen of epigenetic-focused sgRNAs.19 This study found that the loss of anti-silencing function 1a histone chaperone in lung ADC tumor cells led to immunogenicity in the tumor microenvironment and increased sensitivity to anti-programmed cell death-1 (PD-1) immunotherapy.19 Using CRISPR gene knockout for epigenetic modifiers, this study demonstrated a potential therapeutic strategy using gene therapy for KRAS-mutant lung ADC.

The Warburg theory of cancer hypothesizes that cancer cells prefer anaerobic metabolism over aerobic metabolism or oxidative phosphorylation.20 In a recent study on the oxidative phosphorylation gene, pyruvate dehydrogenase E1 alpha subunit (PDHA1), a precursor to oxidative phosphorylation, CRISPR was used to knockout the PDHA1 gene in esophageal cancer cells.21 Plasmid-based CRISPR/Cas9 technology applied to human esophageal cancer cells, which consisted of a plasmid encoding the Cas9 protein and sgRNA, edits the genome inside cells. This strategy led to the deletion of 34 bases in exon 1 of the PDHA1 gene, which led to a terminator codon, resulting in PHDA1 knockout.21 The study of this gene knockout cell line demonstrated the Warburg effect, along with decreased functional tumor suppressor gene p53 and elevated angiogenesis genes.21

Immune Checkpoint Inhibition

Another treatment paradigm in oncology in the past decade has been the use of immune checkpoint inhibitors. The PD-1 and programmed cell death receptor 1 ligand (PD-L1) pathway has formed the basis for an array of immune checkpoint inhibitors that have been FDA approved for a variety of tumor types.22 The expression of PD-L1 on dendritic and tumor cells suppresses antitumor activity and allows cancer to escape the immune system.23 Optimizing T cell activity and function enhances the immune system attack on cancer cells. A recent study administered Cas9 ribonucleoproteins to human T cells to replace specific sequences of the T cell genome.24 Scientists were able to insert targeted nucleotide replacements in T cells at C-X-C chemokine receptor type 4 and PD-1, which has broad applications in the treatment of both HIV and cancer.24

Current checkpoint inhibitor immunotherapy with monoclonal antibodies targets PD-1 on activated T cells and regulatory T cells or PD-L1 on tumor cells to block the inhibitory signaling of T cell activation.25 In a recent study using CRISPR/Cas 9, scientists were able to knock out the PD-1 gene without affecting the viability of primary human T cells in vitro.26 Gene knockout was conducted by electroporation of plasmids encoding the sgRNA-Cas9 DNA.26 This study followed the principle that using 2 sgRNAs to target a gene improves the targeting efficiency and reduces off-target results.27 Gene targeting in T cells is likely to produce a more favorable side effect profile than the immune checkpoint inhibitors, which have immune-related adverse events of colitis, pneumonitis, and transaminitis, among other side effects.28

CAR-T Cell Therapy

Recent developments in another type of immunotherapy, CAR-T therapy, has been shown to have positive response rates in acute lymphoblastic leukemia, chronic lymphoid leukemia, and B-cell lymphoma.29-31 Standard CAR-T therapy is derived from the patient’s own T cells via adoptive T cell transfer, which consists of the ex vivo expansion of the patient’s T cells.32 With the development of CRISPR technology, it is possible to make CAR-T cells from healthy donors in order to maximize CAR-T therapy for a greater number of patients, some of whom may not have enough of their own T cells to harvest for CAR-T therapy. The limitations of this method include graft-versus-host disease (GVHD) and rejection. The T cell receptor is responsible for GVHD because it recognizes antigens as foreign. Using CRISPR knockout, T cell receptors have been silenced in vivo to prevent GVHD in universal CAR-T therapies.33,34 sgRNA and Cas9 were mixed and then electroporated into human T cells isolated from umbilical cord blood.33 The modified CAR-T cells were selected for and expanded and injected back into the patient.33 Although CRIPSR-Cas9 gene editing technologies have enabled the development of universal CAR-T cells in vivo, future studies are warranted in vitro to assess the side effect profile, propensity for GVHD, and efficiency on a larger scale.


In the last decade, gene editing has been revolutionized by CRISPR-Cas9 technology. Most of the research in solid tumors has been in translational research, focusing on mouse models of gene knockout and their applications to future therapies. CRISPR-Cas9 gene knockout has been applied in EGFR- and KRAS-mutated lung cancer and esophageal squamous cell cancer, as described. These applications of CRISPR-Cas9 are summarized in Table 1. Applications of CRISPR to clinical medicine have been demonstrated in hematologic malignancies, specifically acute lymphoblastic leukemia, chronic lymphoid leukemia, and lymphoma.29-31 In current treatment with CAR-T therapy, the patient’s own T cells are used to edit the genome of interest, which is transfused back into the patient. However, many patients, especially children and elderly patients, do not have viable cells for editing. The use of CRISPR to establish universal CAR-T therapy from healthy donors would broaden the availability of CAR-T therapy and allow for more efficient and timely treatment in hematologic malignancies. Additionally, CRISPR has been used successfully in vitro studies of T cell editing, such as CXCR and PD-1 knockout for immune checkpoint inhibition. Potential risks of this method of therapy include GVHD, transfusion reactions, and rejection. Research is ongoing to continue to find improvements in the efficiency and precision of CRISPR gene editsing.

Table 1. Applications of CRISPR in Oncology

Cancer CRISPR mechanism NHEJ/HDR Gene targeted Reference
Lung adenocarcinoma Plasmid NHEJ EGFR-mutated, MEK/ERK signaling 16
Esophageal squamous carcinoma Plasmid NHEJ PDHA1 21
Hematologic malignancies Electroporation of Cas9 ribonucleoproteins NHEJ and HDR CXCR, PD-1 24
Hematologic malignancies, CAR-T therapy Electroporation of Cas9 and invitro transcribed sgRNA NHEJ TCRα subunit constant 32

CAR-T = chimeric antigen receptor T cell; CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats; HDR = homology-directed repair; NHEJ = nonhomologous end joining; TCR = T cell receptor.

Disclosure Statement

The author(s) have no conflicts of interest to disclose.

Author Affiliations

1Drexel University College of Medicine, Philadelphia, PA

2University of California San Francisco Laboratory Medicine, San Francisco, CA

3George Washington University Hospital, Washington, DC 20037

Corresponding Author

Julia Boland ()

Author Contributions

Julia Boland MD participated in acquisition and analysis of the literature and drafting and submission of the final manuscript. Elena Nedelcu MD participated in analysis of the literature and drafting the final manuscript. Both authors have given final approval to the manuscript.

How to Cite this Article

Boland J, Nedelcu E. CRISPR/Cas9 for the Clinician: Current uses of gene editing and applications for new therapeutics in oncology. Perm J 2020;24:20.040. DOI: 10.7812/TPP/20.040


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Keywords: CRISPR, gene editing, oncology


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