Introduction

CRISPR/Cas9 – Clustered Regularly Interspaced Short Palindromic Repeats a revolutionary gene-editing technology, has brought about a significant revolution in biomedical research. This technique enables the correction of genetic errors, as well as the ability to activate or deactivate specific genes within cells and organisms, with remarkable speed, affordability, and simplicity. Its versatility has led to various laboratory applications, including the rapid creation of cellular and animal models, functional genomic screenings, and real-time visualization of the cellular genome (Redman and Watson, 2016).

The extensive possibilities offered by CRISPR/Cas9 go beyond the confines of laboratory research and offer exciting prospects in various clinical domains. Its potential applications encompass gene therapy, combatting infectious diseases such as HIV, and customizing treatments for pediatric patients dealing with conditions like cancer and other illnesses (Plumer & Irfan, 2018).

CRISPR/Cas9, an advanced gene-editing technology, consists of two crucial components: a guide RNA that specifically matches the target gene of interest, and Cas9 (CRISPR-associated protein 9), an endonuclease responsible for creating a double-stranded DNA break. This break enables precise modifications to be made in the genome.

A notable characteristic linked to CRISPR loci is the existence of a preserved sequence known as the "leader," situated upstream of the CRISPR in relation to the direction of transcription (Redman and Watson, 2016). The effectiveness of CRISPR activity relies on the existence of a cluster of CRISPR-associated (Cas) genes, typically located in proximity to the CRISPR region. These cas genes encode the necessary Cas proteins for mounting an immune response (Redman and Watson, 2016).

General Mechanism

The defence mechanism of a bacterium against a bacteriophage is initiated without the bacterium's awareness. In this process, a small Cas protein selectively captures a segment of the phage's DNA and transports it to the CRISPR loci. The captured DNA is then integrated into the CRISPR site. Subsequently, the CRISPR loci are transcribed, leading to the synthesis of crRNA (CRISPR RNA). Together with other proteins, the crRNA recognizes and targets the complementary sequence within the invading phage DNA, resulting in its degradation. The CRISPR/Cas system operates in a similar manner, providing the bacterium with adaptive immunity against phage infections (Sander and Joung, 2014).

CRISPR operates through three distinct stages, namely Acquisition or Adaptation, Expression and Maturation or Biogenesis, and Interference or Targeting. While the process of adaptation remains largely consistent across various classes of CRISPR, the generation of crRNA and the specific targeting mechanism differ among these classes.

Adaptation

The initial stage in the development of a weapon against foreign invading DNA, whether it be viral or plasmid DNA, is adaptation. This process entails the selection of a segment of the invading DNA known as protospacers, which is then incorporated between two neighboring repeats within the CRISPR locus. By integrating the invading DNAs in a specific direction, they become novel CRISPR spacers within a CRISPR array, with repeat sequences acting as dividers. Consequently, this creates a lasting record or memory of the genetic elements that were once invaders.

Expression and maturation phase

After the desired sequences from the invading DNA have been incorporated into the CRISPR loci, transcription takes place, resulting in the formation of pre-CRISPR RNA (pre-crRNA). This transcription occurs from a promoter situated in the AT-rich leader sequence upstream of the CRISPR loci. Subsequently, the pre-crRNA undergoes processing to yield mature crRNAs. Each crRNA comprises a transcribed spacer sequence that is fused to a partial repeat sequence (Sander and Joung, 2014).

Interference phase

The crRNA associates with Cas proteins to form a complex. Within this complex, the crRNA aligns with the protospacer region of the invading DNA through base pairing. Ultimately, the Cas proteins within the crRNA-Cas complex execute a targeted cleavage of the invading DNA at the protospacer site, which corresponds to the complementary sequence of the crRNA spacer.

[picture source: Molecular Cell 54, April 24, 2014 [5]]

Types of CRISPR/Cas systems

There are several types of CRISPR systems that have been discovered and studied by Harrison & Wildonger (2014). Here are some of the main types:

1. Type I: This type of CRISPR system is characterized by a large multi-subunit complex called Cascade (CRISPR-associated complex for antiviral defense). It uses a crRNA (CRISPR RNA) to target and cleave invading DNA.

2. Type II: The Type II CRISPR system is the most well-known and widely used system. It utilizes a single Cas protein called Cas9, along with a guide RNA (gRNA), which is a synthetic RNA molecule that directs Cas9 to specific DNA sequences for editing.

3.Type III: Type III CRISPR systems employ a multi-subunit complex called Csm (CRISPR-associated complex for antiviral defense) to target and cleave RNA molecules instead of DNA. They can also have additional Cas proteins involved in the process.

4. Type IV: Type IV CRISPR systems are the least characterized among the known types. They are characterized by a unique set of Cas proteins and have been found in some bacteria and archaea.

5. Type V: The Type V CRISPR system utilizes a single Cas12 protein (previously known as Cpf1). Cas12 functions similarly to Cas9, cleaving DNA at target sites specified by a guide RNA (crRNA).

6. Type VI: The Type VI CRISPR system is also known as the CRISPR-Cas13 system. It targets and cleaves RNA rather than DNA. The Cas13 protein is guided by a CRISPR RNA (crRNA) to specifically degrade RNA sequences.

Applications

CRISPR technology has revolutionized the field of molecular biology and has a wide range of applications (Barrangou & Doudna, 2016). Some of the notable applications of CRISPR include:

Gene Editing: CRISPR-Cas9 is widely used for precise and efficient gene editing in various organisms, including plants, animals, and human cells. It allows scientists to modify specific DNA sequences, either by introducing targeted mutations or by inserting new genetic material.
Disease Treatment: CRISPR holds promise for treating genetic disorders by correcting disease-causing mutations. It can be used to edit the DNA of affected cells and potentially cure genetic diseases like sickle cell anemia, cystic fibrosis, and muscular dystrophy.
Agriculture: CRISPR-based gene editing techniques can be applied to improve crop traits, such as disease resistance, drought tolerance, and nutritional content. It offers a more precise and efficient way to develop genetically modified crops with desired characteristics.
Drug Discovery: CRISPR can be used to create disease models in the laboratory, allowing researchers to better understand the mechanisms of diseases and develop new drugs. It enables the targeted disruption or modification of specific genes associated with diseases, providing valuable insights into their function.
Livestock Improvement: CRISPR technology can be employed to improve livestock breeding by introducing desirable traits and removing harmful ones. It has the potential to enhance animal health, productivity, and welfare.
Biotechnology: CRISPR is being utilized in various biotechnological applications, such as developing biosensors, creating biofuels, producing bioactive compounds, and modifying industrial microorganisms for improved production processes.
Disease Diagnosis: CRISPR-based diagnostic methods, such as CRISPR-based detection assays and CRISPR-based nucleic acid amplification tests (CRISPR-NAT), offer rapid and sensitive detection of pathogens and genetic variations associated with diseases.
Conservation Biology: CRISPR can aid in conservation efforts by helping to preserve endangered species or restore damaged ecosystems. It can be used to modify invasive species, combat pests, or enhance the resilience of endangered species.

Conclusion

CRISPR technology has emerged as a groundbreaking tool in the field of genetic engineering. Its ability to precisely edit DNA sequences has opened up new possibilities in medicine, agriculture, biotechnology, and conservation. With the potential to treat genetic diseases, enhance crop traits, develop new drugs, and aid in conservation efforts, CRISPR holds immense promise for addressing some of the most pressing challenges facing humanity. However, it is important to proceed with ethical considerations, responsible use, and appropriate regulation to ensure the safe and beneficial application of CRISPR in various domains.

References:

Redman, M., King, A., Watson, C., & King, D. (2016). What is CRISPR/Cas9?. Archives of Disease in Childhood-Education and Practice, 101(4), 213-215.

Plumer, B., Barclay, E., Belluz, J., & Irfan, U. (2018). A simple guide to CRISPR, one of the biggest science stories of the decade. VOX Media.

Barrangou, R., & Doudna, J. A. (2016). Applications of CRISPR technologies in research and beyond. Nature biotechnology, 34(9), 933-941.

Harrison, M. M., Jenkins, B. V., O’Connor-Giles, K. M., & Wildonger, J. (2014). A CRISPR view of development. Genes & development, 28(17), 1859-1872.

Sander, J. D., & Joung, J. K. (2014). CRISPR-Cas systems for genome editing, regulation and targeting. Nature biotechnology, 32(4), 347.

Pickar-Oliver, A., & Gersbach, C. A. (2019). The next generation of CRISPR–Cas technologies and applications. Nature reviews Molecular cell biology, 20(8), 490-507.