Genome editing is a kind of genetic engineering in which scientists are able to change an organism’s DNA. This technology allows specific genes to be inserted, replaced, modified, and deleted. One type of gene editing is known as the CRISPR-Cas9 system (or clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9). The CRISPR-Cas9 system has proven to be of great benefit to the scientific community, as the system is often seen as the most simplest, accurate, versatile, and inexpensive genome editing tool.
CRISPR and Cas genes are fundamental to adaptive immunity in certain bacteria and archaea, allowing the organisms to respond and eliminate invading genetic material. CRISPR, a group of DNA sequences in bacteria, contains snippets of DNA from viruses that have attacked the bacteria. These snippets, called CRISPR arrays, enable the bacteria to detect and eventually destroy DNA of similar virus invaders during future attacks. These sequences form the basis of the CRISPR/Cas system – a prokaryotic immune system.
Three types of CRISPR mechanisms have been currently found, of which type II is the most studied. In this type of CRISPR, exogenous DNA from viruses and plasmids are cut into small pieces and combined into the CRISPR gene in a series of short repeats. The gene is then transcripted and translated into mini RNAs known as crRNA or CRISPR RNA. These crRNAs are used as a guide for Cas9 nuclease which targets invading DNA by complimentary sequencing. Cas9 is an enzyme that acts a pair of molecular scissors, and is able to cut through two strands of DNA in a genome. Usually cas or CRISPR-associated protein take form in small clusters near to the CRISPR sequences. In type II systems, cas9 functions in the transcripting of crRNA as well as in the destruction of targeted DNA. Cas9’s ability to perform in these steps is due to the presence of two nuclease domains that is located at the amino terminus and mid-region of the enzyme. During the breakdown of targeted DNA, a twenty-nucleotide long sequence within crRNA transcript designates the sites at which the two nuclease domains cut both DNA strands. The simplistic technique seen in the type II CRISPR system enables it to be multifaceted in means of genome editing. Its versatile capacity was recognized by the Doudna and Charpentier Labs in 2012. A simplified version of the type II CRISPR system was created by developing a single guide RNA (sgRNA) in place of crRNA. This replication proved to be as effective as the crRNA fuelled Cas9 in guiding targeted gene modifications. In a typical lab procedure, the sgRNA binds to the Cas9 enzyme and navigates to the targeted DNA sequence in a genome. The Cas9 enzyme cuts the DNA at the target point and finally, scientists use the cell’s DNA repair mechanism to insert, delete, or modify fragments of genes.
Following its debut in 2012, the CRISPR Cas9 genome editing method has been widely used. This is mainly due to the system’s only necessity of modification of the crRNA to change the particular target DNA contrasting to other editing tools such as TALENs and zinc finger who require change in the entire protein-DNA-interface. This method has gained success in targeting genes of many organisms’ cells such as zebrafish, C. elegans, Drosophila, monkeys, Xenopus tropicalis, yeats, rabbits, pigs, rats, mice, and even humans. However, it will require much more laboratory testing as well as time before it is regularly used on humans.
Currently, research is being done on genome editings’ potential use in the treatment and prevention of human disease. To determine whether this method is safe on people, tests are being accomplished to understand the effects of CRISPR Cas9 on a wide variety of diseases including monogenetic illnesses like hemophilia, cystic fibrosis, and sickle-cell, using isolated cells and animal models. Parameters such as target efficiency, or the percentage of the desired mutation achieved, is essential in developing a safe, accurate method to genome editing. Cas9 efficiency has proven to be more precise than other methods such as TALENs or ZFNs. For example, specially developed ZFNs and TALENs were reported to have only attained a range of 1% to 50% efficiency in human cells, while the CRISPR Cas9 system achieved up to 70% in certain plants and zebrafish. Furthermore, a group of scientists using paired sgRNAs to target a single gene at the same time, were able to increase efficiency to 78% in a single-cell mouse embryo, successfully accomplishing germline editing.
In the future, genetic editing might hold the answer to diseases such as cancer, mental illness, human immunodeficiency virus (HIV), and heart disease. However, the use of genome editing, such as CRISPR, in human genes, has sparked controversy. Proposed modifications involving germ-line cells. has brought up debates concerning ethical implications. Changes to the egg and sperm cells not only affect certain tissues, but they are passed down from generation to generation. Because of issues on the safety and morals of germline genome editing, it has been banned from several countries, limiting gene modifications to that of somatic cells.
The future of the CRISPR Cas9 genome editing system holds great potential to reinvent the way we approach diseases. With the system’s simplicity, efficiency, and versatility, accuracy for transmission of genetic material is becoming more and more developed each day, serving to continually redefine its capabilities. Scientific breakthroughs to catastrophic diseases seem fathomable within the promising future of the editing system. With the use of CRISPR Cas9, cell and molecular research progress, with hopes of creating a brighter future for generations to come. The system’s usage in genome editing is perhaps only limited by the imagination.
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