Monday Article #60: Cystic Fibrosis and CRISPR/Cas9
Cystic fibrosis (CF) is a prevalent genetic disease caused by a recessive allele, resulting in an impairment of mucus production by epithelial cells. CF progressively affects various bodily systems, including the respiratory, digestive, reproductive, and sweat gland systems. This disease arises due to mutations in the Cystic Fibrosis Transmembrane (CFTR) protein, responsible for transporting chloride ions out of the cell. As a result of these mutations, chloride ions accumulate within the intracellular spaces, disrupting the balance of salt and water on various body surfaces, such as the lungs. The dysfunction of CFTR protein leads to the thickening and stickiness of mucus, giving rise to the characteristic symptoms of cystic fibrosis.
While the body possesses natural mechanisms, including DNA repair systems, to correct certain DNA mutations, some mutations persist and are transmitted during the process of protein synthesis.
In recent years, the development of gene editing technologies has provided a potential solution to address genetic disorders like cystic fibrosis. This article focuses on the application of CRISPR/Cas9, a revolutionary gene editing tool, in the treatment of cystic fibrosis.
How does the mutation occur?
The main cause of Cystic Fibrosis is a mutation in the CFTR protein. There are multitudes of ways in which that mutation could occur. However, the most common mutation is the deletion of an amino acid, called phenylalanine at codon 508 (known as ΔF508).
To comprehend this mutation more, we must first understand that DNA is degenerate, meaning that a single amino acid can be coded for by multiple different codons.
In the ΔF508 CFTR sequence, the bases C, T, and T are deleted. Thus, another base T substitutes the previously deleted base C, resulting in base deletion and base substitution to form an ATT codon that codes for an amino acid called Isoleucine.
Figure 1. The abnormal DNA base sequence of ΔF508 CFTR sequence.
The Mechanism of CRISPR/Cas9
CRISPR, an acronym for Clustered Regularly Interspaced Short Palindromic Repeats, represents specific repetitive sequences found in the DNA of bacteria. Interestingly, these repeats are derived from fragments of viral DNA. Bacteria utilise these sequences as a molecular surveillance system to recognize and combat viral infections. Cas9, an enzyme found in bacteria, plays a pivotal role in the CRISPR system by acting as a pair of "molecular scissors" capable of cleaving DNA.
In the context of treating cystic fibrosis, the utilisation of CRISPR/Cas9 involves three essential components: the Cas9 nuclease, guide RNA, and repair template containing the correct gene sequence.
Figure 2. The three components of CRISPR mechanism
The guide RNA serves as the key element in directing the Cas9 nuclease to the specific target site on the DNA that requires modification. Upon binding to the guide RNA, the Cas9 nuclease unzips the DNA and examines whether the guide RNA sequence aligns with the DNA. If there is a match, the Cas9 nuclease cleaves the DNA, generating a break in the DNA strands.
Subsequently, the mutation is corrected through a repair process utilising the repair template. The repair template replaces the mutant sequence with a normal sequence, restoring the genetic integrity. Following this correction, the repair template dissociates, and the intact strand of DNA utilises the repaired strand as a template to fill in the remaining gaps. Ultimately, both strands of DNA harbour the corrected gene sequence, thereby enabling the CFTR protein to regain normal functionality.
Challenges and Limitations of CRISPR/Cas9 Implementation
Despite the groundbreaking research conducted by Jennifer Doudna and Emmanuelle Charpentier, leading to their Nobel Prize for the development of CRISPR technology, several challenges hinder the widespread implementation of CRISPR/Cas9 in clinical settings. Healthcare professionals and scientists remain cautious about its clinical application due to several concerns.
One significant limitation is the delivery of CRISPR/Cas9 components to the target cells within the body. Currently, researchers primarily rely on viral vectors to transport Cas9 DNA into cells, especially for in vivo applications. However, the persistent presence of viral vectors can lead to prolonged Cas9 expression, potentially causing unintended modifications in the genome over extended periods.
The issue of off-target effects represents another safety concern associated with CRISPR/Cas9. Although Cas9 is designed to cleave specific DNA sequences, there is a possibility of Cas9 making unintended cuts in non-targeted regions of the genome. Such off-target effects can introduce additional mutations, including those associated with an increased risk of cancer. Consequently, researchers are actively working towards enhancing the specificity of CRISPR/Cas9 to mitigate the risk of off-target effects.
CRISPR/Cas9 holds immense promise as a gene editing tool for the treatment of genetic disorders such as cystic fibrosis. However, its clinical implementation faces challenges related to the delivery of CRISPR components and the risk of off-target effects. Ongoing research and advancements in CRISPR technology aim to overcome these limitations and pave the way for the safe and effective application of CRISPR/Cas9 in the treatment of cystic fibrosis and other genetic disorders, such as cancer, high cholesterol, HIV, and Huntington's disease.
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This article was prepared by Christabelle Johneva Lee