CRISPR-Cas9: Revolutionizing Genetic Engineering

Introduction

CRISPR-Cas9, often simply referred to as CRISPR, is a groundbreaking technology that has transformed the field of genetic engineering. This powerful tool enables scientists to edit genes with unprecedented precision, efficiency, and resilience. Since its introduction, CRISPR-Cas9 has opened up new possibilities in research, medicine, and agriculture.

History

The journey of CRISPR-Cas9 began with the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) in the DNA of bacteria. These sequences were initially identified as part of the bacterial immune system, which protects against viral infections. In 2012, Jennifer Doudna and Emmanuelle Charpentier published a seminal paper demonstrating how the CRISPR-Cas9 system could be adapted for precise gene editing in other organisms [1]. This discovery earned them the Nobel Prize in Chemistry in 2020.

Procedure

Researchers have developed refined protocols for CRISPR-Cas9 applications, such as generating large deletions, introducing point mutations, and creating knockout mutants [2,3]. These protocols often involve designing guide RNA sequences, constructing CRISPR/Cas9 constructs, transfecting target cells, and selecting for desired mutations. Additionally, advancements like the all-in-one CRISPR-Cas9 system have simplified genome editing processes by streamlining the cloning pipeline.

The CRISPR-Cas9 genome editing procedure involves several key steps that enable precise modifications in the target DNA. The CRISPR-Cas9 system consists of two key components: the Cas9 enzyme and a synthetic guide RNA (sgRNA). Here’s a step-by-step overview of how it works:

1. Designing the Guide RNA (gRNA)

Initially, a cloning step is undertaken to create a plasmid that encodes the Cas9 enzyme along with a synthetic guide RNA (sgRNA) designed to target specific genomic sites [4]. The first step in the CRISPR-Cas9 process is to identify the specific DNA sequence within a gene that you want to edit. This target sequence is usually around 20 base pairs long. The target sequence should be unique to minimize off-target effects (unintended edits in other parts of the genome).

Scientists design a synthetic guide RNA (sgRNA) that is complementary to the target DNA sequence. The sgRNA consists of two parts:

• CRISPR RNA (crRNA): This portion is complementary to the target DNA sequence.
• Trans-activating crRNA (tracrRNA): This helps the crRNA bind to the Cas9 enzyme.

In practice, these two RNA components are often fused into a single synthetic guide RNA for simplicity.

2. Introducing the CRISPR-Cas9 Complex

The CRISPR-Cas9 complex consists of the gRNA and the Cas9 enzyme. Cas9 is a nuclease, meaning it can cut DNA.

The delivery of the CRISPR-Cas9 components can be achieved through various methods, such as electroporation, transfection, or in utero electroporation, depending on the target organism or cell type [3,5]. Moreover, this can be done through various methods, such as:

• Viral Vectors: Modified viruses that can deliver the CRISPR components into cells. The most commonly employed viral vectors for delivering CRISPR/Cas are adeno-associated viruses (AAVs) [6], adenoviral vectors (AdVs) [7], and lentiviral vectors (LVs) [8].
• Lipid Nanoparticles: Lipid nanoparticles (LNPs) are becoming a desirable nonviral delivery system for CRISPR-mediated genome editing because of their minimal immune response and versatile applicability. [9].
• Microinjection: The CRISPR-Cas components are delivered into the zygote through direct microinjection into the cytoplasm [10].

3. Binding and Cutting

Once inside the cell, the sgRNA binds to the complementary DNA sequence within the genome. The Cas9 enzyme, guided by the sgRNA, is then directed to the exact location where the cut is to be made. Cas9 requires a short sequence adjacent to the target DNA, known as the protospacer adjacent motif (PAM), to bind and cut. The presence of a PAM sequence (usually "NGG" in the case of the most commonly used Cas9 from Streptococcus pyogenes) is critical for Cas9 activity. Cas9 makes a double-strand break (DSB) in the DNA at the target site. This break is a clean cut through both strands of the DNA helix, which initiates the cell’s natural DNA repair mechanisms. [11]

4. DNA Repair

The sgRNA guides the Cas9 nuclease to the desired DNA sequence, inducing double-strand breaks that trigger DNA repair mechanisms, such as classical non-homologous end joining (NHEJ), homologous recombination (HR), microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA) [12]. One of the primary repair mechanisms is Non-Homologous End Joining (NHEJ), which quickly rejoins the broken DNA ends. However, this process is error-prone and can lead to insertions or deletions (indels), which may disrupt the gene and knock out its function and is often used when the goal is to disable a gene [13,14].

If scientists want to introduce specific changes (e.g., correcting a mutation), they can provide a DNA template along with the CRISPR-Cas9 complex. The cell uses this template to repair the break via Homology-Directed Repair (HDR), incorporating the desired changes into the genome. HDR is more precise than NHEJ, but it is also less efficient and requires the cell to be in a specific phase of the cell cycle. These repair pathways lead to the introduction of specific modifications in the genome, such as gene deletions, mutations, or insertions [15]

5. Verification and Validation

After the CRISPR-Cas9 process is complete, scientists must verify that the intended edits were made correctly and that there are no off-target effects. This is done through sequencing the DNA around the target site and screening for any unintended modifications.

Scientists often conduct functional assays to confirm that the gene edit has the desired biological effect. For example, if the goal was to knock out a gene, they would check if the gene is indeed non-functional. Sanger or short-read sequencing is frequently used to validate genome editing [16].

Applications

The CRISPR-Cas9 system has been successfully applied in various organisms, including plants, animals, and parasites, showcasing its broad utility[17-20]. This technology has been instrumental in generating knockout models, introducing somatic mutations, and facilitating gene corrections in -induced pluripotent stem cells and mammalian cell lines [21-23]. Moreover, CRISPR-Cas9 has been employed in high-throughput screening techniques, allowing for the investigation of gene function and biological processes on a genome-wide scale [2,3].

Applications of CRISPR-Cas9 across various fields are explained in following points.

• Medicine: It holds promise for treating genetic disorders such as cystic fibrosis, sickle cell anemia, and muscular dystrophy. Clinical trials are underway to explore its potential in cancer therapy[24,25].
• Agriculture: CRISPR is used to develop crops with improved traits, such as disease resistance, drought tolerance, and enhanced nutritional content [26].
  
• Research: It enables scientists to create model organisms for studying diseases, understand gene functions, and develop new therapies[27].

Conclusion

CRISPR-Cas9 is a revolutionary tool that has transformed genetic engineering. Its ability to precisely edit genes has far-reaching implications for medicine, agriculture, and scientific research. As the technology continues to evolve, it promises to unlock new possibilities and address some of the most pressing challenges in biology and medicine.

References

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