In recent years there has been astounding development in medicine, and diseases that were initially incurable are now treatable. The most effective technique to treat an illness and improve health is through alterations in the genetic sequence. Every living organism consists of innumerable cells containing a core of vital information which determines every aspect of that living organism. This indispensable core of information is known as the genome. Changes in the genomic material of an organism result in changes in the organism’s phenotype, disease resistance, and disease-causing ability.
1- What is gene editing?
Genome editing, also known as gene editing, features a list of techniques through which scientists can alter an organism’s DNA. Through these revolutionary technologies, scientists can add, delete or change the sequence of nucleotides in the organism’s DNA. There are several methods of performing genome editing.
2- What is gRNA?
gRNA is shorthand for Guide RNA and refers to a short chain of synthetic molecules of RNA that scientists use in the CRISPR system of gene editing.
3- What is the CRISPR system?
The CRISPR system is a highly sophisticated form of gene modification. It consists of a sequence of almost 20 bp nucleotides, which connects with a specific target site on a molecule of DNA. This site of attachment is known as the spacer sequence. The molecule of synthetic gRNA is an anchor for the protein-based endonuclease enzyme Cas. After binding with Cas, it directs it to the target site on the molecule of DNA to initiate gene editing. Hence, the two essential parts of CRISPR-based genome editing are the gRNA and the CRISPR-associated endonuclease (Cas). The CRISPR-Cas system is quicker, more affordable, more precise, and more effective compared to other methods of gene editing; hence, it has created a lot of curiosity in the scientific community.
4- What was the inspiration for the CRISPR system?
The inspiration for the CRISPR-Cas9 system came from bacteria. Bacteria have a naturally present gene editing system that they use as a defense mechanism. When a virus infects a bacterium, the bacterium grabs pieces of the viral DNA and integrates them into its own genome in a specific pattern. This creates special segments of DNA known as CRISPR arrays. Through the CRISPR arrays, the bacterium remembers the virus that attacked it and other similar types. Hence, when the same virus or a closely related one attacks again, the bacterium assembles RNA segments using the CRISPR arrays. These RNA segments identify and bind to specific sites on the viruses’ DNA. The bacterium then splices the viral DNA using Cas9 or a similar protease, killing the virus.
5- How do scientists use gRNA in the CRISPR system?
Researchers learned from this defense mechanism in bacteria to develop a process of editing DNA. They created a short chain of synthetic gRNA that binds to a specific location on a cell’s DNA, similar to the action of RNA segments that bacteria develop from the CRISPR arrays. This guide RNA also has a scaffold for attachment with the Cas9 enzyme. When scientists introduce the gRNA into host cells, it recognizes the target site on the cell’s DNA, and the Cas9 enzyme snips the DNA at the intended location, similar to the DNA splicing in bacteria. Cas9 is the enzyme scientists use most often, although other enzymes like Cpf1 are also applicable. Once scientists break up the DNA, they use the host cell’s machinery to alter it by adding or removing genetic material.
6- What is the purpose of the gRNA in the CRISPR system?
Gene editing via the CRISPR system has taken the medical industry by storm. Researchers and doctors are looking into it with immense interest and are keen on using it to prevent and treat human diseases. Currently, they are experimenting with genome editing on animal cells in research labs to understand the pathophysiology of various diseases. Scientists are working tirelessly to decipher whether they can use this procedure safely on humans. Scientists are testing this innovative technique in laboratories to discover better treatment options for several diseases. The primary focus is on single-gene disorders such as cystic fibrosis, hemophilia, and sickle cell disease. Researchers also hope this technique will bear promising results for treating and preventing more complex diseases like HIV, mental illnesses, cancer, and heart disease.
7- What else is gRNA used for?
Recombinant gRNA has several uses apart from the CRISPR system. Scientists use a combination of gRNA and similar reagents in labs to perform diagnostic tests like multiplex PCR, classic PCR, cloning, whole-genome sequencing, development of primers, cell lines, and recombinant plasmid DNA, and mismatch assay for DNA mutagenesis, modification, and studies related to gene expression.
8- How do scientists design gRNA?
Scientists use CRISPR design tools to create multiple gRNA sequences effectively. They modify the wild-type gRNA segments in laboratories to determine their efficiency in altering DNA and formulating treatments. Scientists use a DNA template and custom crRNA combined with the scaffold tracrRNA segment to design and create gRNA in vitro or in vivo.
The specificity of the CRISPR-Cas9 system for the target site varies depending on the 20 nucleotide sequence at the 5’-end of the gRNA strand. Combining the crRNA and tracrRNA results in the formation of gRNA, after which it attaches to the target site and makes a ribonucleoprotein (RNP) complex with the Cas9 protein. Before scientists start designing a gRNA strand, it is necessary to guarantee that the GC range of the gRNA falls between 40-80%, and the gRNA sequence should not be more than 17-24 base pairs long to prevent any effects off-target effects.
Although, so far, experiments with gRNA have shown promising results, there is still a lot of uncertainty regarding the efficacy and effects of this revolutionary technique. Moreover, there is no way to choose the most suitable gRNA for the results you desire. However, some of the elements that affect its accuracy include the strategy the scientists use to create gRNA, such as synthetic methods, lentiviral delivery techniques, or in vitro transcription. Another factor to bear in mind is the arrangement of chromatin, which will determine the ability of gRNA to reach the target site.
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