Nature review: How CRISPR gene editing is transforming cancer research, diagnosis and treatment

Since its inception, the CRISPR gene editing system has become a powerful tool for studying cellular function
.

It has a major impact on our understanding of cancer biology and continues to drive new discoveries that accelerate the development of cancer diagnostics and treatments

The impact of CRISPR on cancer biology research

The impact of CRISPR on cancer biology research

Precision medicine strategies for cancer rely on the discovery of genetic mutations that promote cancer growth, and CRISPR gene editing technology can rapidly and efficiently generate gene knockouts that modulate endogenous gene expression and replicate cancer-related genomic changes
.

Due to the simplicity and efficiency of CRISPR technology, it has become routine to generate gene knockout mouse models

▲Using CRISPR technology to generate different types of cancer models (Image source: Reference [1])

Perhaps the biggest impact CRISPR technology has had on cancer research is CRISPR screening, the review authors noted
.

This screen uses a library of guide RNAs (gRNAs) targeting different genes in the genome to systematically knock out any gene in a cell line or organoid, and then observe the effect of the gene knockout on cancer cell growth or drug response

Another important application of CRISPR in cancer research is tracking lineage changes in cancer cells
.

One of the hallmarks of cancer is its heterogeneity, with cancer cells accumulating genetic variations that lead to clones of cells with different characteristics

At present, researchers have developed a variety of CRISPR-based tracking strategies that can find different cell clones in mixed cell populations containing multiple cell clones and track their dynamic changes over time
.

CRISPR technology can introduce unique barcodes into cells that can be used to mark cancer cell lineages

▲ Different CRISPR strategies for tracing the clonal lineage of cancer cells (Image source: Reference [1])

CRISPR Applications in Cancer Diagnostic Development

CRISPR Applications in Cancer Diagnostic Development

Early detection of cancer provides the best chance of curing cancer, and CRISPR technology can help develop more sensitive molecular diagnostic tools to aid in the early detection of cancer
.

CRISPR molecular diagnostic systems based on Cas12 and Cas13 have been used to detect cancer-related genetic mutations from patient tumor tissue biopsies

In addition, the CRISPR system can be used to precisely cut DNA fragments in specific regions of the genome.
Compared with traditional random genome fragmentation, this method can enrich the DNA fragments of interest, which can be combined with next-generation gene sequencing.
, gene mutations on specific genes can be found in very small sample sizes
.

This technique is currently being evaluated in clinical trials for the detection of p53 mutations in ovarian cancer

CRISPR applications in cancer therapy

CRISPR applications in cancer therapy

In cancer treatment, one of the main applications of CRISPR technology is to engineer immune cells to generate anti-cancer immunotherapies
.

Multiple research teams have shown that targeting PD-1 expression in T cells using CRISPR gene editing can increase the anticancer activity of T cells

In addition, CRISPR gene editing can be used to knock out human leukocyte antigen (HLA) on the surface of endogenous T cell receptors and T cells from healthy donors when producing allogeneic "universal" CAR-T cell therapies , thereby reducing the risk of immune rejection and graft-versus-host disease caused by allogeneic cell infusion
.

Moreover, using CRISPR/Cas9 technology, the CAR-expressing sequence can also be specifically inserted into the gene locus of the T cell receptor alpha constant region (TRAC) of the cell, resulting in the consistent expression of the CAR
.
In vitro and in mouse models, CAR-T cells generated by this approach exhibited enhanced anticancer activity compared to conventional CAR-T cells
.

▲Various ways of CRISPR-engineered T cells (Image source: Reference [1])

Using CRISPR gene editing to directly target the genetic variants that drive cancer, beyond in vitro engineered T-cell therapy, is an attractive, but also very difficult, challenge
.
In theory, CRISPR gene editing can directly correct the genetic mutation that drives the cancer, or kill cancer cells that produce a specific genetic mutation, which can inhibit tumor growth
.
However, this strategy requires overcoming multiple hurdles, including the delivery of tumor-specific therapies, and the need for high-efficiency gene editing
.

Current preclinical studies have identified several strategies for tumor-specific gene editing
.
For example, CRISPR gene-editing systems that target oncogene fusions can selectively target tumor cells while disrupting the gene mutations that promote tumor growth
.
Another preclinical study put the transcription of the CRISPR-Cas13a system under the control of the NF-κB transcription factor.
Since NF-κB is overactivated in a variety of cancers, this strategy can specifically express the CRISPR-Cas13a system in cancer cells , Knock down the expression of oncogenes to achieve the effect of inhibiting the growth of cancer cells
.

▲Using NF-κB to control the expression of CRISPR-Cas13a system, tumor-specific degradation of oncogenes (Image source: Reference [4])

In terms of delivery technology, lipid nanoparticles (LNP) have achieved great success in delivering 2019-nCoV mRNA
.
The use of LNP to deliver Cas9-encoding mRNA and gRNA has effectively targeted the mitotic-essential gene PLK1 in proof-of-concept studies, reaching 70% in vivo gene editing efficiency in a mouse model of glioblastoma, causing apoptosis and inhibiting Tumor growth by 50% and improved survival by 30%
.
Antibodies targeting tumor-specific antigens conjugated on the surface of LNPs also successfully drove scattered tumors to selectively absorb LNPs, improving tumor-specific editing efficiency
.

Taken as a whole, these preclinical studies show the potential of in vivo CRISPR gene editing for the treatment of cancer, although much effort is still required to translate CRISPR in vivo gene editing into viable clinical-stage anticancer therapies
.

Limitations and Prospects

Limitations and Prospects

Although CRISPR has broad uses in cancer biology, the review authors note that further development of this technology, especially in clinical treatment, still needs to overcome several limitations
.
DNA double-strand breaks caused by Cas enzymes can trigger inadvertent loss of DNA fragments and, in some cases, drive chromosome fragmentation (chromothripsis), which affects normal cell function
.
DNA double-strand break damage caused by CRISPR technology may stimulate the p53 signaling pathway, resulting in cell death or selection of cells with reduced p53 function
.
In addition, the off-target editing potential of CRISPR systems has been a concern for researchers
.

However, while these potential limitations are important limitations driving the further development of CRISPR technology, scientists have now developed tools to detect and minimize the occurrence of these events
.
The authors say they may not significantly hinder the clinical application of CRISPR technology
.
In the next 5-10 years, CRISPR technology will truly enter the stage of clinical-stage therapy, and work in CAR-T therapy and other immune cell engineering heralds their role in the treatment of cancer
.

In scientific research, CRISPR technology has begun to address many of the fundamental questions of cancer.
By characterizing the role of individual genes in cancer cell behavior, it will provide the foundation for building the next generation of immunotherapies, uncovering the role of noncoding regions and regulatory elements in tumorigenesis and many other fields
.
CRISPR technology has been and will be one of the key tools in our understanding and treatment of human cancers
.

References:

References:

[1] Katti et al.
, (2022).
CRISPR in cancer biology and therapy.
Nature Reviews Cancer, https://doi.
org/10.
1038/s41568-022-00441-w

[1] Katti et al.
, (2022).
CRISPR in cancer biology and therapy.
Nature Reviews Cancer, https://doi.
org/10.
1038/s41568-022-00441-w

[2] Zhang et al.
, (2022).
Application of the CRISPR/Cas9-based gene editing technique in basic research, diagnosis, and therapy of cancer.
Molecular Cancer, https://doi.
org/10.
1186/s12943-021 -01431-6

[2] Zhang et al.
, (2022).
Application of the CRISPR/Cas9-based gene editing technique in basic research, diagnosis, and therapy of cancer.
Molecular Cancer, https://doi.
org/10.
1186/s12943-021 -01431-6

[3] Gong et al.
, (2022).
CRISPR/Cas-Based In Vitro Diagnostic Platforms for Cancer Biomarker Detection.
Analytical Chemistry, https://doi.
org/10.
1021/acs.
analchem.
1c02533

[3] Gong et al.
, (2022).
CRISPR/Cas-Based In Vitro Diagnostic Platforms for Cancer Biomarker Detection.
Analytical Chemistry, https://doi.
org/10.
1021/acs.
analchem.
1c02533

[4] Gao, et al.
, (2020).
A new tool for CRISPR-Cas13a-based cancer gene therapy.
Mol.
Ther.
Oncolytics, https://doi.
org/10.
1016/j.
omto.
2020.
09.
004

[4] Gao, et al.
, (2020).
A new tool for CRISPR-Cas13a-based cancer gene therapy.
Mol.
Ther.
Oncolytics, https://doi.
org/10.
1016/j.
omto.
2020.
09.
004