Editor’s recommendation: “Nature Reviews Cancer” published a review article entitled “CRISPR in biology and therapy”, which systematically reviewed the role of CRISPR systems in cancer The latest advances in 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 and accelerate the development of cancer diagnosis and treatment.

Recently, “Nature Reviews Cancer” published a review article entitled “CRISPR in cancer biology and therapy”, which systematically reviewed the application of CRISPR system in cancer research, Recent advances in diagnosis and treatment.

The impact of CRISPR on cancer biology research

Precision cancer medicine Strategies rely on the discovery of genetic mutations that promote cancer growth, and CRISPR gene editing technology can quickly 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. Furthermore, tissue-specific cancer models can be generated by selectively introducing all components of the CRISPR gene editing system into specific somatic cells.

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.

Using CRISPR technology to generate different types of cancer Model

The authors of this review point out that the biggest impact of CRISPR technology on cancer research may be CRISPR screening.

This screen systematically knocks out any gene in a cell line or organoid using a library of guide RNAs (gRNAs) targeting different genes in the genome, and then observes the knockout Effects on cancer cell growth or drug response after removal.

Another important application of CRISPR in cancer research is the tracking of 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 gives scientists a more comprehensive understanding of tumorigenesis.

Currently, researchers have developed a variety of CRISPR-based tracking strategies to discover and track different cell clones in mixed cell populations containing multiple cell clones Dynamic changes over time.

CRISPR technology can introduce unique barcodes into cells 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.

Tracking different CRISPR clones in cancer cells Strategy

CRISPR in the development of cancer diagnosis

Early detection of cancer offers the best chance of curing cancer, and CRISPR technology can help develop more sensitive molecular diagnostic tools to aid in early detection of cancer. CRISPR molecular diagnostic systems based on Cas12 and Cas13 have been used to identify cancer-associated genetic mutations from patient tumor tissue biopsies.

They emit fluorescent signals by cleaving an RNA sequence carrying a fluorescent reporter protein after finding a specific oncogene mutation sequence.

During the COVID-19 pandemic, this technology was used to produce rapid and sensitive tests for the novel coronavirus.

The same platform can be used to generate highly sensitive and personalized cancer discovery and surveillance systems.

In addition, the CRISPR system can be used to precisely cut DNA fragments in specific regions of the genome.

Compared with traditional random genome fragments, this method can enrich DNA fragments of interest and can be combined with next-generation gene sequencing. In the United States, genetic mutations in specific genes can be found in very small sample sizes. The technology is currently being evaluated in clinical trials for the detection of p53 mutations in ovarian cancer.

CRISPR in cancer therapy

in cancer therapy , one of the main applications of CRISPR technology is to engineer immune cells to produce anticancer immunotherapies.

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. These candidate therapies are already in clinical trials.

In addition, CRISPR gene editing can be used to destroy human leukocyte antigen (HLA) receptors on the surface of endogenous T cells, taking advantage of healthy donations”universal” CAR-T cell therapy in which the patient produces allogeneic T cells, thereby reducing the risk of immune rejection and graft-versus-host disease, which are caused by allogeneic cell infusion.

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 cells, thereby To make CAR expression consistent.

CAR-T cells generated by this method exhibited higher anticancer activity than conventional CAR-T cells in vitro and in mouse models.

CRISPR can transform a variety of T cells Ways

In addition to in vitro engineered T cell therapy, using CRISPR gene editing to directly target cancer-causing genetic variants is an attractive but also very difficult challenge .

Theoretically, 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.

However, this strategy needs to overcome multiple hurdles, including the delivery of tumor-specific therapies, and the need for efficient gene editing.

Current preclinical studies have identified several tumor-specific gene editing strategies. For example, CRISPR gene-editing systems that target oncogene fusions can selectively target tumor cells while destroying genetic mutations that promote tumor growth.

Another preclinical study placed transcription of the CRISPR-Cas13a system under the control of the NF-κB transcription factor.

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 inhibition effect on cancer cell growth.

Using NF-κB to control CRISPR-Cas13a Systematic expression for tumor-specific degradation of oncogenes

In terms of delivery technology, lipid nanoparticles (LNP) have achieved great success in delivering 2019-nCoV mRNA. great success.

Current usage provides Cas9-encoding mRNA and gRNA effectively targeting PLK1, a gene essential for mitosis, and in a proof-of-concept study, in vivo gene editing efficiency reached 70% gel A mouse model of plasmoblastoma, resulted in apoptosis and inhibition of tumor growth and a 50% increase in survival by 30%.

Antibodies targeting tumor-specific antigens bound to the surface of LNPs also successfully drove scattered tumors to selectively uptake LNPs, improving tumor-specific editing efficiency.

Overall, these preclinical studies show the potential of in vivo CRISPR gene editing in cancer treatment, although translating in vivo CRISPR gene editing into a viable clinical-stage anticancer therapy Much effort is still required.

Limitations and Prospects

Although CRISPR has Widespread application, but the authors of this review 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 unintended loss of DNA fragments and, in some cases, drive chromosome breakage (chromothripsis), which affects normal cellular function.

The 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. Furthermore, 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 reduce 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 really enter the clinical stage, and CAR-T therapy and other immune cell engineering work herald their use in cancer treatment effect.

In scientific research, CRISPR technology has begun to solve many fundamental problems in cancer. 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. CRISPR technology has been and will be one of the key tools in our understanding and treatment of human cancer.

Reference: https://www.nature.com/articles/s41568-022-00441-w

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