"Nature Review" Application of CRISPR in Cancer Research, Diagnosis and Treatment

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

Recently, the journal "Nature Reviews Cancer" published a review article entitled "CRISPR in cancer biology and therapy", which systematically reviewed the latest progress of the CRISPR system in cancer research, diagnosis and treatment

The impact of CRISPR on cancer biology research

Cancer precision medicine strategies 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

For example, using CRISPR to edit the Tet2, Runx1, Dnmt3a, Nf1, and Smc3 genes in hematopoietic stem cells can stimulate acute myeloid leukemia

Delivering CRISPR to the liver, pancreas or lung can rapidly generate cancer models with complex phenotypes

The review authors note that the biggest impact of CRISPR technology on cancer research may be CRISPR screening

The 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 knockout on cancer cell growth or drug response

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

A hallmark of cancer is its heterogeneity, with cancer cells accumulating genetic variation, resulting in cell clones with distinct characteristics

Understanding the heterogeneity within tumors and tracking the generation and evolution of new clones has given scientists a more comprehensive understanding of tumorigenesis

Researchers have developed a variety of CRISPR-based tracking strategies to discover different cell clones in mixed cell populations containing multiple cell clones and track their dynamics over time

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

The latest CRISPR recording systems are able to introduce markers in the genome at specific times, and by analyzing these different markers, lineage relationships between different cancer cell clones can be constructed

Different CRISPR strategies for tracking cancer cell clone lines

Application of CRISPR in the Development of Cancer Diagnostics

Early detection of cancer offers the best chance of curing it, and CRISPR technology could help develop more sensitive molecular diagnostic tools to aid in the early detection of cancer
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CRISPR molecular diagnostic systems based on Cas12 and Cas13 have been used to identify cancer-associated genetic mutations from patient tumor tissue biopsies
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They emit fluorescent signals by cleaving an RNA sequence carrying a fluorescent reporter protein after finding a specific oncogene mutation sequence
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During the COVID-19 virus pandemic, this technology was used to produce rapid and sensitive tests for the novel coronavirus
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The same platform can be used to generate highly sensitive personalized cancer discovery and surveillance systems
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In addition, the CRISPR system can also be used to precisely cut DNA fragments in specific regions of the genome
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Compared to traditional random genome fragments, this method enriches DNA fragments of interest and can be combined with next-generation gene sequencing
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In the United States, genetic mutations in specific genes can be found in very small sample sizes
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The technology is currently being evaluated in clinical trials for the detection of p53 mutations in ovarian cancer
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CRISPR in Cancer Therapy

In cancer treatment, one of the main applications of CRISPR technology is to engineer immune cells to produce anticancer immunotherapies
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Multiple research teams have demonstrated that targeting PD-1 expression in T cells using CRISPR gene editing can increase the anticancer activity of T cells
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These candidate therapies are already in clinical trials
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In addition, CRISPR gene editing can be used to destroy human leukocyte antigen (HLA) receptors on the surface of endogenous T cells, using a "universal" CAR-T cell therapy that produces allogeneic T cells from healthy donors, thereby reducing immune rejection Reactions and the risk of graft-versus-host disease, which are caused by allogeneic cell infusion
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In addition, using CRISPR/Cas9 technology, the CAR expression sequence can also be specifically inserted into the gene locus of the T cell receptor alpha constant region (TRAC) of the cell, so that the expression of the CAR is consistent
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In vitro and in mouse models, CAR-T cells generated by this method exhibited higher anticancer activity than conventional CAR-T cells
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The many ways CRISPR can engineer T cells

In addition to engineering T-cell therapies in vitro, using CRISPR gene editing to directly target cancer-causing genetic variants presents an attractive but also very difficult challenge
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In theory, CRISPR gene editing can directly correct the genetic mutation that causes cancer, or kill cancer cells that develop a specific genetic mutation, thereby inhibiting tumor growth
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However, this strategy needs to overcome multiple hurdles, including the delivery of tumor-specific therapies, and the need for efficient gene editing
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Current preclinical studies have identified several tumor-specific gene editing strategies
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For example, CRISPR gene-editing systems that target oncogene fusions can selectively target tumor cells while destroying genetic mutations that promote tumor growth
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Another preclinical study placed transcription of the CRISPR-Cas13a system under the control of the NF-κB transcription factor
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Since NF-κB is over-activated in various cancers, this strategy can specifically express the CRISPR-Cas13a system in cancer cells, knock down the expression of oncogenes, and achieve the effect of inhibiting the growth of cancer cells
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Using NF-κB to control the expression of the CRISPR-Cas13a system for tumor-specific degradation of oncogenes

In terms of delivery technology, lipid nanoparticles (LNPs) have achieved great success in delivering 2019-nCoV mRNA
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The current state of use provides Cas9-encoding mRNAs and gRNAs that efficiently target PLK1, a gene essential for mitosis, and in a proof-of-concept study achieved 70% in vivo gene editing efficiency in a mouse model of glioblastoma, resulting in apoptosis and Inhibition of tumor growth and 50% increased survival by 30%
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Antibodies targeting tumor-specific antigens bound to the surface of LNPs also successfully drove dispersed tumors to selectively uptake LNPs, improving tumor-specific editing efficiency
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Overall, these preclinical studies show the potential of in vivo CRISPR gene editing in cancer therapy, although significant efforts are still required to translate in vivo CRISPR gene editing into viable clinical-stage anticancer therapies
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Limitations and Prospects

Despite the broad application of CRISPR in cancer biology, the authors of this review note that further development of the technology, especially in clinical treatment, still needs to overcome several limitations
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DNA double-strand breaks caused by Cas enzymes can trigger unintended loss of DNA fragments and, in some cases, drive chromosome breakage (chromothripsis), which affects normal cellular function
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DNA double-strand break damage caused by CRISPR technology may stimulate the p53 signaling pathway, leading to cell death or cell selection with reduced p53 function
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Furthermore, the off-target editing potential of CRISPR systems has been a concern for researchers
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However, despite these potential limitations being important limitations driving the further development of CRISPR technology, scientists have now developed tools to detect and reduce the occurrence of these events
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The authors say they may not significantly hinder the clinical application of CRISPR technology
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In the next 5-10 years, CRISPR technology will truly enter the clinical stage, and work on CAR-T therapy and other immune cell engineering heralds their role in cancer treatment
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In scientific research, CRISPR technology has begun to solve many fundamental problems in cancer
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By characterizing the role of individual genes in cancer cell behavior, it will provide the basis for building the next generation of immunotherapies, revealing the roles of noncoding regions and regulatory elements in tumorigenesis and many other areas
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CRISPR technology has been and will be one of the key tools in our understanding and treatment of human cancer
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