Introductory Chapter: CRISPR Technology

1. Introduction

The clustered regularly interspaced short palindromic repeats (CRISPR) was originally derived from bacteria fighting against foreign genetic material, such as plasmid or viral DNA. This is an adaptive immunity generated in the bacteria infected with bacteriophage. Traditionally, bacteria will have a memory of the DNA they have invaded, and when DNA with the same sequence enters the bacteria again, an acquired immune response will be generated to break down the foreign DNA. CRISPR consists of multiple, short, and direct repeats of DNA sequences, each repeat containing a series of bases accompanied by about 30 bases called spacer DNA. These spacers are short DNA fragments from plasmids or bacteriophages. When the host encounters this particular plasmid or bacteriophage again, it will recognize the foreign DNA by complementation with CRISPR RNA (crRNA). After crRNA binds to complementary foreign DNA, the Cas9 protein (nuclease) breaks down and destroys the invading DNA or RNA. The mechanism is that single-stranded guide RNA (sgRNA) interacts with Cas9, and the combination of sRNA and Cas9 will guide the endonuclease activity to the region adjacent to the protospacer sequence (PAM). After the sgRNA recognizes a specific DNA sequence, the bound Cas9 will cut 3 nucleotides upstream of the PAM (NGG) of the positive and negative DNA strands, forming a double-stranded break with a blunt end. Because CRISPR technology is becoming more mature and stable, it has been successfully applied in genetic editing, diagnosis, and medicine for years.

2. Genetic editing

Gene cloning is to select a specific target gene to manipulate the genetic traits of an organism and then use molecular biology methods to modify the target gene. The modified genome is put into the target gene and recombined into an expression vector and finally transformed into a suitable host cell for a large number of expressions. The main production processes, include vector gene cleavage, target gene cutting, recombinant vector, transformed host, recombinant vector replication, and host cell culture, including gene transfer between the same species and different species to improve or generate new animals, plants, and microorganisms.

Traditional gene cloning methods use restriction enzymes to cut specific restriction sites in the genome. After selecting a gene, the cutting position is not accurate enough, screening is time-consuming, labor intensive, and expensive. Genetic editing of organisms based on CRISPR technology has the advantages of high efficiency, accuracy, speed, and economy, compared with traditional methods. Genetic editing has revolutionized biological research through the new ability to precisely edit the genomes of living organisms. Recently, various genetic tools have been explored for engineering simple and complicated genomes. The CRISPR/Cas9 system has widely been used in genetic editing because of its high efficiency, ease of use, convenience, and accuracy. It can be used to add desirable and remove undesirable genes simultaneously in a single event. Additionally, many newly emerging CRISPR/Cas systems, such as base editor, xCas9, Cas12a (Cpf1), and Cas13 are also considered. The scientific community has already used this technology to modify human cells, animals, plants, or microorganisms. Transgenic animals and plants or engineered microorganisms are used in basic research, such as viruses, bacteria, yeasts, protozoa, plants, mice and human cells. Many literatures related to CRISPR technology have been published in the past few years, and genetic editing is currently the most successful and extensive application [1, 2].

3. Diagnosis

The traditional methods (e.g., isolation and identification, nucleic acid and antigen detection, and specific antibody detection.) used to perform diagnosis of microbe infection in humans or contamination in food are not only time-consuming, labor-intensive, and expensive but also require sophisticated equipment and the professionals. Therefore, it is necessary to develop a new intelligent and rapid diagnostic method that does not need to rely on professional equipment and personnel. CRISPR technology has been introduced into the field of rapid nucleic acid detection for the development of new medical detection tools and reagents, bringing breakthroughs to existing testing and diagnostic technologies. For example, the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) system for CRISPR/Cas12a (Cpf1) enables analysis of cells, blood, saliva, urine, and feces to detect genetic mutations, cancer, and antibiotic resistance and can be used to diagnose bacterial and viral infections. We take the detection of human papillomavirus (HPV), Zika virus, dengue virus, and SARS-CoV-2 as examples to show the potential of CRISPR technology as a diagnostic tool.

3.1 Human papillomavirus

In 2018, Jennifer Anne Doudna’s team at the University of California, Berkeley, used the CRISPR/Cas12a (Cpf1) system to cut the target’s double-stranded DNA (dsDNA) and found that the Cas12a nuclease would be activated and nonspecifically cleave single-stranded DNA (ssDNA), which deliver the CRISPR/Cas12a system and nonspecific ssDNA fluorescent labeling (FQ-labeled reporter) into cells [3]. Once the target dsDNA is detected, the CRISPR/Cas12a system will be activated and the fluorescent reporter gene will also be degraded to release a fluorescent signal. In a previous study, they demonstrated that CRISPR can be a tool for diagnosing viral infections in vitro. The DETECTR can rapidly and accurately detect HPV infections in patient specimens. The detection rate of HPV16 and HPV18 infection is 100% (25/25 agreement) and 92% (23/25 agreement), respectively, and HPV16 and HPV18 are known to be the most dangerous subspecies causing cervical cancer [3].

3.2 Zika virus and dengue virus

In 2018, Feng Zhang’s team in the Broad Laboratory of Massachusetts Institute of Technology (MIT) combined isothermal preamplification with Cas13 nuclease to detect single-stranded RNA (ssRNA) and ssDNA tool-specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) to deliver a variety of enzymes and fluorescent reporter genes to cells [4]. If the CRISPR system finds the target gene, the corresponding Cas13 will activate the cleavage enzyme and specifically cut the corresponding fluorescent reporter gene, which releases fluorescent signals. The SHERLOCK can be used to detect ssRNA viruses, such as Zika virus and dengue virus in human specimens, such as saliva. After the introduction of a variety of bacterial Cas13 nucleases from different genera, such as LwaCas13a and PsmCas13b, SHERLOCK was upgraded to SHERLOCK version 2 (SHERLOCKv2). Because different Cas 13 nucleases show different degrees of “preference” for different RNA sequences, SHERLOCKv2 sensitivity is increased by 3.5 times, compared with the original SHERLOCK [4].

3.3 COVID-19 (SARS-CoV-2)

In 2020, Chinese researchers summarized the latest progress on COVID-19 detection (SARS-CoV-2 infection) based on various CRISPRs, including CRISPR/Cas9, CRISPR/Cas12, and CRISPR/Cas13, which are being developed as a fast, accurate, and portable diagnostic method, showing the potential of CRISPR to be applied in the diagnosis of COVID-19 and other emerging infectious diseases [5]. Compared with the polymerase chain reaction (PCR) assay and DNA sequencing methods, this new method can more rapidly identify the pathogens of emerging infectious diseases and facilitate timely treatment. While ideal detection reagents are characterized by being fast, reliable, inexpensive, and convenient, these emerging diagnostics still require careful testing and clinical validation to ensure their functionality [5].

In 2020, Ackerman et al. proposed a platform called CARMEN (specific high-sensitivity enzymatic unlocking and an extension of SHERLOCK), which could detect a range of pathogen infections, including the new coronavirus that causes COVID-19 [6]. The detection mixture contains sgRNA, Cas13, and a fluorescently labeled reporter RNA. The fluorescent molecule is attached to the reporter RNA and does not emit light. SgRNA can recognize a specific target nucleic acid (DNA or RNA) sequence. If the CRISPR/Cas13 complex recognizes the target nucleic acid sequence, Cas13 will be activated to cleave the reporter RNA and generate fluorescence, thereby detecting a specific virus infection in the specimen [6].

In 2020, Mammoth Biosciences and GSK announced the development of a CRISPR-based SARS-CoV-2 detection platform DETECTR, which can rapidly (less than 40 minutes) and accurately detect SARS-CoV-2 from nasal swab RNA extracts from examinees [7]. The method was validated using reference samples and clinical samples from patients, including 36 patients with COVID-19 and 42 patients with other viral respiratory infections in the United States (US). They found that the test results had a positive predictive concordance of 95% and a negative predictive concordance of 100%, demonstrating that this is a visible and rapid detection method and has the potential to replace the most widely used quantitative real-time reverse transcription polymerase chain reaction assay (qRT-PCR) [7]. This detection platform is characterized by being fast, easy to operate, portable, and completely disposable. It can identify SARS-CoV-2 RNA through a simple nasal swab and does not require professionals, laboratories, and instruments. This DETECTR can obtain test results within 20 minutes and detect a variety of infectious diseases. GSK submitted the entire testing platform to the US FDA for evaluation in 2020 and promoted it as a point-of-care testing tool for hospitals and clinics. The US FDA has conducted an emergency use authorization (EUA) review of the platform. The platform is expected to provide over-the-counter tests for the general public to use at home in the near future.

4. Medicine

CRISPR can be used for drug discovery and screening successfully and significantly facilitate the pharmaceutical development. However, human therapeutics based on CRISPR for which direct disease treatment was only attempted or under clinical trial due to the concerns of safety, efficacy, and ethics, including inheritance disease, viral infections, neurodegenerative diseases, metabolic diseases, and cancer. Currently, the most perspective fields for the treatment based on CRISPR are precision medicine and gene therapy.

4.1 Precision medicine

The most sophisticated and sensitive field of CRISPR development should be therapeutic. Because human therapy is related to life, health, and human rights, it requires the highest technical level, and all critical issues must be considered. The most stringent regulatory requirements and restrictions are performed around the world. The most important application of CRISPR technology in human medicine should be precision medicine, also known as personalized treatment. This concept was first proposed by the National Research Council of the US in 2011. Human individuals will show different traits due to genetic differences, and the symptoms and severity of the disease will also vary. The same treatment methods are used for different individuals with the same disease, but they may have different therapeutic effects. Therefore, it is often necessary to have different treatment strategies depending on individual differences. In addition to the patient’s description of symptoms and routine examinations (e.g., blood test, X-ray examination, and ultrasound examination) that are used in traditional methods, precision medicine also includes biomedical tests, such as genetic testing, protein testing, and metabolic testing. For precision medicine, they analyze personal data (e.g., gender, height, weight, race, past medical history, family medical history, and test results.) through the human genetic database to select the most suitable strategy, and drugs for patients to maximize the therapeutic effect and minimize side effects. CRISPR technology can accurately perform gene editing and has the potential to correctly repair gene mutations, which can be applied to the clinical treatment of diseases, thus becoming precision medicine.

4.2 Gene therapy

Although CRISPR has the potential to be directly used in human therapy, most of them are only at the stage of basic research or animal experiments, and few are actually conducted clinical trials or product launches because of lack of specificity, fear of causing mutations, and difficulty in delivery tool selection. It is complicated and there are many options for treatment. For most diseases, the direct use of CRISPR- based treatment may not be effective and even lead to aggravating the disease if you rashly skip conventional treatment methods. However, there are exceptions, especially if the main cause of diseases is due to defective genes. For congenital genetic diseases, traditional therapy usually only alleviates the symptoms but not completely cure the disease, and a complete cure requires the modification or repair of genes. In these special cases, the safety and ethics concerns of treatment based on CRISPR are less, thereby the compassionate use is possible. Therefore, many scientists are focusing on developing CRISPR as a tool for gene therapy.

In 2021, the United Kingdom and New Zealand researchers revealed the drug NTLA-2001 developed from CRISPR/Cas9 can treat the rare disease “familial amyloid polyneuropathy” [8]. This disease is mainly due to gene mutation, which accumulates misfolded transporter (transthyretin, TTR). The main function of NTLA-2001 is to reduce the concentration of TTR in serum. After completing the preclinical trials in vitro and in vivo, the team conducted a clinical phase 1 trial to evaluate the safety and efficacy of a single escalating dose of NTLA-2001 on patients, with a total of six subjects [8]. Animal studies in preclinical studies have shown that the TTR gene can be permanently knocked out after a single dose. In contrast to the results of the Phase 1 clinical trial, patients were assessed for safety within the first 28 days after the infusion of the drug, and few adverse events were found. On day 28, serum TTR protein concentrations were found to be reduced by an average of 52% from baseline in patients receiving the 0.1 mg/kg dose, compared with an 87% reduction at the 0.3 mg/kg dose. In a small group of patients with hereditary ATTR and polyneuropathy, existing research results show that NTLA-2001 effectively reduces the concentration of pathogenic TTR protein in serum by targeting TTR gene through CRISPR, while only mild adverse events occur [8].

5. Conclusion

The use of CRISPR technology for genetic editing, diagnosis, drug development, and screening is extensive and less controversial because it is not directly related to human therapeutics. CRISPR has the potential to be used for the treatment of diseases, including genetic disease, viral infections, neurodegenerative disorders, metabolic diseases, cancer and regenerative medicine; however, there are still considerable safety, efficacy, and ethics concerns for the treatment based on CRISPR. Many pharmaceutical companies and biotechnology companies have begun to produce various medical products using CRISPR technology. Only few cell therapy and gene therapy products based on CRISPR have been approved on the market, and some are still under clinical trials. Currently, the conventional treatments have been tested and proven to be effective for most diseases. With the advancement of medical technology and the development of new drugs, many diseases that were considered incurable in the past have been treated well now. Therefore, patients should use conventional treatment first and consider using CRISPR-based therapy, while the disease cannot be controlled or existing drugs cannot cure the disease. According to the current testing results, most of the treatments based on CRISPR are still in the stage of accumulating experience, and more clinical data are still needed to prove their effectiveness and safety. It belongs to medium and high-risk medical behaviors. Gene therapy, using CRISPR, is likely to be a suitable method to be tested for the treatment of inherited diseases because inherited diseases can be cured only when the defective gene is modified. Before patients receive direct treatment based on CRISPR, genetic testing should be performed to confirm suitability, consultation with professional physicians must be completed, and the evaluation of whether the study meets the regulations of compassionate use is required to ensure the welfare of patients.