n REVIEW ARTICLE
CRISPR/Cas9 for the Clinician: Current uses of gene editing and
applications for new therapeutics in oncology
Julia Boland, MD1,3; Elena Nedelcu, MD2
E-pub: 12/09/2020
ABSTRACT
Precise genomic editing has given rise to treatments in pre-
viously untreatable genetic diseases and has led to revolutions in
treatment for cancer. In the past decade, the discovery and de-
velopment of clustered regularly interspaced short palindromic
repeats (CRISPR) technologies has led to advances across medi-
cine 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 thera-
peutic purposes in different oncologic tumors, and review novel
treatment paradigms using CRISPR in immuno-oncology, in-
cluding checkpoint inhibitors and chimeric antigen receptor T cell
therapy.
INTRODUCTION
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.
erefore, 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 de-
rived from the natural adaptive immune system of bac-
teria. Current applications of research in CRISPR have
been in studying gene knockout in cancers, editing mu-
tated genes, and engineering T cells for chimeric antigen
receptor T cell (CAR-T) therapy. is review summarizes
CRISPR techniques and highlights the applications of the
technology to cancer therapeutics.
Background
e 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
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Perm J 2020;24:20.040
https://doi.org/10.7812/TPP/20.040
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
e 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 e CRISPR-Cas9-sgRNA complex then edits
the genome of interest. ere are many Cas proteins;
however, Cas9 is the most efficient and widely used.7 e
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 con-
tribute to cancer risk through carcinogenesis and chromo-
somal 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 po-
tentially 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
ere are various methods of delivery and carriers of the
CRISPR-Cas9 system. e delivery of sgRNA and Cas9
Author Affiliations
1
Drexel University College of Medicine, Philadelphia, PA
2
University of California San Francisco Laboratory Medicine, San Francisco, CA
3
George Washington University Hospital, Washington, DC 20037
Corresponding Author
Julia Boland (julialindsayboland@gmail.com)
Keywords: CRISPR, gene editing, oncology
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REVIEW ARTICLE
CRISPR/Cas9 for the Clinician: Current uses of gene editing and applications for new therapeutics in oncology
Figure 1. Pathway for dsDNA break in CRISPR. NHEJ = Non-homologous end
joining; HDR = Homology-directed repair.
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 e delivery mechanisms vary on
efficiency and gene editing errors.
Review
Translational 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 car-
cinoma, 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 ade-
nocarcinoma. Patients with EGFR mutations can be sus-
ceptible to targeted therapy with gefitinib and erlotinib,
which are first-generation competitive, reversible inhibitors
of the EGFR-tyrosine kinase. ese 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 e 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 Ap-
proximately 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 produc-
tion 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 im-
mune responsiveness to checkpoint inhibitor therapy with
epigenetic modification is one method of improving response
to therapy. Epigenetic modifications consist of DNA base
2 ·
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methylation and histone modification, which affects gene
expression.18 A study of KRAS-mutant lung ADC con-
structed an in vivo CRISPR screen of epigenetic-focused
sgRNAs.19 is 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 in-
creased sensitivity to anti-programmed cell death-1 (PD-1)
immunotherapy.19 Using CRISPR gene knockout for epige-
netic modifiers, this study demonstrated a potential therapeutic
strategy using gene therapy for KRAS-mutant lung ADC.
e Warburg theory of cancer hypothesizes that cancer
cells prefer anaerobic metabolism over aerobic metabolism or
oxidative phosphorylation.20 In a recent study on the oxi-
dative phosphorylation gene, pyruvate dehydrogenase E1
alpha subunit (PDHA1), a precursor to oxidative phos-
phorylation, 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. is 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 e
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.
e 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 ap-
proved for a variety of tumor types.22 e 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 Sci-
entists were able to insert targeted nucleotide replace-
ments 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 mono-
clonal antibodies targets PD-1 on activated T cells and reg-
ulatory 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 is study followed the principle that using 2
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CRISPR/Cas9 for the Clinician: Current uses of gene editing and applications for new therapeutics in oncology
Table 1. Applications of CRISPR in Oncology
Cancer CRISPR mechanism
Lung adenocarcinoma Plasmid
Esophageal squamous carcinoma Plasmid
Hematologic malignancies Electroporation of Cas9
ribonucleoproteins
Hematologic malignancies, CAR-T therapy Electroporation of Cas9 and invitro
transcribed sgRNA
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.
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 trans-
aminitis, 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. e
limitations of this method include graft-versus-host
disease (GVHD) and rejection. e 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 e modified CAR-T cells were
selected for and expanded and injected back into the
patient.33 Although CRIPSR-Cas9 gene editing tech-
nologies 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.
CONCLUSIONS
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
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REVIEW ARTICLE
NHEJ/HDR Gene targeted Reference
NHEJ EGFR-mutated, MEK/ERK signaling 16
NHEJ PDHA1 21
NHEJ and HDR CXCR, PD-1 24
NHEJ TCRα subunit constant 32
esophageal squamous cell cancer, as described. ese ap-
plications 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 ge-
nome of interest, which is transfused back into the patient.
However, many patients, especially children and elderly pa-
tients, do not have viable cells for editing. e 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 hema-
tologic malignancies. Additionally, CRISPR has been
used successfully in vitro studies of T cell editing, such as
CXCR and PD-1 knockout for immune checkpoint in-
hibition. 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. v
Disclosure Statement
The author(s) have no conflicts of interest to disclose.
Authors’ 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:
https://doi.org/10.7812/TPP/20.040
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