Application of CRISPR/Cas9 gene editing technology

Gene editing is an emerging genetic engineering technology that can modify specific target genes in the genome of organisms. Gene editing can efficiently perform site-directed genome editing, so it has shown great application prospects in gene research, gene therapy and gene breeding.

To date, CRISPR/Cas9 has been widely used in the scientific community as an efficient gene editing tool. Gene editing is a technique that precisely modifies genome sequences to induce insertions, deletions, or base substitutions in the genome. Many diseases are accompanied by changes in gene expression in the body, especially some hereditary diseases caused by single gene mutations, and gene editing technology is expected to control the occurrence of diseases at the gene level.

Traditional gene editing tools mainly include the second-generation TALEN and the third-generation CRISPR technology, both of which use nucleases containing DNA recognition binding domain and DNA cleavage domain, but there are problems such as low target recognition rate, high cost, high off-target probability and complex structure, so the development of third-generation CRISPR technology has been promoted.

▲The development of gene editing technology (Huang et al., 2021).

CRISPR-related gene editing technology is one of the most popular biological tools at present. Since 2013, CRISPR technology research has exploded, and tens of thousands of CRISPR-related articles have been published, as shown in Figure 1.

Fig. 1 Timeline of major events in the development of CRISPR/Cas technology and representative Cas9 variants (Li et al., 2023).

Today we are going to talk about CRISPR/Cas9, a genome editing technology that has the advantages of low cost, easy operation and high efficiency. We will briefly explain the theoretical background and main applications of CRISPR/Cas9 technology, hoping to inspire your research.

Introduction to CRISPR/Cas9 technology

In the late 80s and early 90s of the 20th century, researchers observed a DNA sequence in bacterial and archaeal genomes, known as clustered regularly interspaced short palindromic repeat repeats (CRISPR). With the progress of research, it is recognized that the CRISPR/Cas system is an immune defense system developed by bacteria and archaea in the long-term evolution process to resist the invasion of foreign plasmids and phages, and it can excise foreign nucleic acids through gRNA localization Cas enzymes. There are three main types of CRISPR/Cas systems (type I, type II, and type III), but they differ in the molecular mechanisms by which nucleic acid recognition is achieved. In contrast to types I and III, type II systems rely only on RNA-guided single proteins for specific DNA recognition and cleavage, making them the most commonly used genome editing tools. The CRISPR/Cas9 system is a type II CRISPR/Cas system, derived from Streptococcus pyogenes SF370, which can be gene edited by single-guide RNA (sgRNA) sequences and Cas9 endonucleases, and is a novel genome editing technology based on RNA-guided endonucleases.

The CRISPR/Cas9 system consists of two main components: the Cas9 protein and guide RNA (gRNA). The Cas9 protein contains RuvC1 and HNH-like nuclease domains, which have the function of cleaving double-stranded DNA, resulting in a break in the double-stranded DNA. The PAM motif is the recognition site of Cas9, and the PAM motif of Cas9 is 5′-NGG. Cas9 cleaves double-stranded DNA at the 3rd base upstream of the PAM motif. crRNA and tracrRNA are formed by local base pairing, which is a small RNA molecule designed to bind to a specific target sequence in the target DNA. Jennifer Doudna and Emmanuelle Charpentier fused crRNA and tracrRNA into a single RNA, which it called sgRNA. Once the sgRNA binds to its target, it recruits the Cas9 protein, which acts as a molecular scissor to cleave the DNA at the target site.

The technical principle of CRISPR/Cas9 is to use a gRNA complementary to the target sequence to guide the Cas9 protein to recognize and cleave the specific target DNA, so as to cause a DNA double-strand break and produce a specific DNA double-strand break (DSB). After DSB is formed, the double-strand break is repaired by the cell’s own non-homologous end joining (NHEJ) or homologous recombination repair (HDR), and finally the genomic genetic modification such as target gene knockout, knock-in, and base editing is realized. However, NHEJ is prone to errors, so after the formation of DSB, the homologous sequence of the target site can be introduced into the cell as a repair template, and then HDR can be performed to achieve precise gene editing.

▲Schematic diagram of type II CRISPR system-mediated DNA double-strand breaks (Cong L and Zhang F., 2015).

Gene editing in mammalian cells

In 2013, Feng Zhang et al. used human 293T cells to integrate tracrRNA, pre-crRNA, RNase III, and Cas9 into them, and added their respective promoters and two nuclear localization signals (NLS) to ensure structural access to the nucleus (Cong et al., 2013). In another paper published in the same year, the researchers constructed crRNA-tracrRNA fusion transcripts, i.e., sgRNAs and scaled the crRNA down to 20 bp (Mali et al., 2013). These studies are of great significance, both confirming the role of CRISPR in mammalian cells and simplifying the CRISPR gene editing system, opening up more possibilities for the use of CRISPR. In the months that followed, scientists rapidly achieved gene editing in animals such as mice, fruit flies, and rats, as well as plants such as rice and wheat, and Schwank et al. used CRISPR/Cas9 technology to correct disease-causing mutations in the intestinal stem cells of two CFTR mutant patients.

The first clinical trial of CRISPR/Cas9 technology was the injection of CRISPR/Cas9 gene-edited T cells back into patients, which was the world’s first injection of gene-edited cells into humans, which demonstrated the feasibility and safety of the clinical application of gene editing technology, and is of great significance for promoting the clinical application of gene editing technology (Lu et al., 2020).

Although CRISPR/Cas9 has successfully cured some diseases caused by point mutations by cleaving the double strands for rerepair, the inefficiency and uncertainty of this method limit its application. The researchers believed that the treatment of genetic diseases should correct the mutated base rather than excise it to allow random recombination, laying the foundation for the invention of single-base gene editing technology (CBE, ABE) and PE, the editing principle of which is shown in Figure 2.

Fig.2 Schematic diagram of single-base editing and PE editing mechanisms (Wang et al., 2022).

CRISPR technology and oncology treatment

Malignancy is a disease with high morbidity and mortality, and the standard treatment is surgical resection, radiotherapy, and chemotherapy; However, the latter two treatments can have serious side effects. One of the main purposes of CRISPR/Cas9 screening in oncology is to identify genotype-specific weaknesses, targeted deletion of these genes can reduce the viability of cancer cells, thus providing a strategy for discovering potential therapeutic targets, and combining CRISPR screening with drugs can provide insight into how tumors respond to drug treatments.

Adoptive cell therapy (ACT) is an immunotherapy that uses immune cells, especially T cells, to fight tumor cells. Tumor-infiltrating lymphocyte (TIL) therapy was one of the first ACT. However, ACT is subject to a number of practical limitations, including difficulty in isolating enough qualified T cells from patients with advanced cancer and infants. There are two main ACT approaches currently being studied: chimeric antigen receptor (CAR)-T cell therapy and genetically modified T cell receptor (TCR)-T cell therapy (Figure 3).

Figure 3 The three main approaches of ACT and the application of CRISPR in tumors (Wang et al., 2022).

1. CAR-T cell therapy

CAR is a recombinant antigen receptor that can alter the specificity and function of T lymphocytes, resulting in a powerful anti-tumor response. CARs consist of single-stranded variable fragments fused to transmembrane and intracellular signaling regions, typically with one or two costimulatory domains (Figure 4). Fig. 4 Structure of first- to third-generation CARs (Wang et al., 2022).

Following the FDA’s approval of two autologous CAR-T cell therapies in 2017, multiple clinical trials have shown that CAR-T cell therapies are effective for a variety of hematologic and non-hematologic malignancies, and most clinical trials currently use autologous CAR-T cells, which are isolated from patients, genetically edited to express CAR structures, and then expanded in vitro and infused back to patients, but this method is not only expensive, but differences in the number and quality of patient T cells may seriously affect efficacy. At the same time, the efficacy of CAR-T cell therapy in solid tumors remains to be studied, and the simplicity and accessibility of CRISPR/Cas9 gene editing can address many of these limitations, so the current research efforts are focused on using the traditional CRISPR/Cas9 system or novel editors to edit CAR-T cells so that they can be directly used to target negative regulators of T cell function and direct therapeutic transgenes to specific genomic loci. and to generate reproducible, safe, and effective allogeneic universal CAR-T cell products for personalized tumor immunotherapy (Figure 5).

Figure 5 Summary of CRISPR/Cas9 editing strategies to generate the most effective and widely used CAR-T cell products (Dimitri et al., 2022).

2.TCR-T cell therapy

Clinical trials have shown that CAR-T cell therapy has limited efficacy in solid tumors, mainly due to the lack of tumor-specific antigens, tumor heterogeneity, and suppressive immuno-tumor microenvironment. Similar to CARs, T cells can be modified with defined TCRs to make them respond to specific tumor antigens. TCR-T cells have shown great potential in immunotherapy, as CARs can recognize target antigens such as glycolipids and cell surface proteins, but there are far more tumor-specific antigens located within cells and presented by MHC molecules than tumor-specific antigens on the cell surface, which can be recognized by TCR-T cells, so TCR-T cell therapy may be a potential treatment for solid tumors. However, TCR-T target antigen profiling is limited and requires MHC molecules for antigen presentation, and tumor cells can achieve immune escape by down-regulating the expression level of MHC molecules, which has become a major disadvantage of this treatment.

One of the main problems caused by the introduction of exogenous TCRs into T cells is the pre-existing endogenous TCRs on recipient T cells, as the introduction of exogenous TCRs results in the formation of new reactive TCR dimers consisting of introduced TCR chains paired with endogenous TCRs α or β chains, which are potentially pathogenic and hinder the expression of transgenic TCRs (tgTCRs), and the competition between endogenous TCRs and tgTCRs for CD3 molecules further limits the expression of the tgTCR complex, This set of problems can lead to off-target effects, which can lead to reduced therapeutic efficacy and deadly autoimmunity. The use of gene editing tools to mediate the disruption of endogenous TCR α and β-chain genes is a strategy to eliminate competition and has demonstrated feasibility in primary T cells (Figure 6). In 2020, the Phase 1 human clinical trial reported the use of multiple CRISPR/Cas9 editing to target TRAC, TRBC, and PDCD1 in T cells to reduce TCR mismatches and improve anti-tumor immune responses, and the introduction of synthetic tumor-specific TCR transgenes to recognize tumor cells, and the engineered T cells injected into patients persist in vivo for up to 9 months, preliminarily suggesting that the combination of TCR transfer and genome editing may lead to the development of more effective and safer tumor immunotherapies.

Fig.6 Structure of TCR dimer and application of CRISPR/Cas9 in TCR-T cell therapy (Wang et al., 2022).