CRISPR/Cas9 is a gene editing method that relies on an endonuclease and eukaryotic DNA repair mechanisms. Short for clustered and regularly interspaced short palindromic repeats1, CRISPR originally existed as a natural immunity of bacteria against viral infections. Since the discovery of CRISPR, researchers have been able to identify, isolate and modify the CRISPR machinery and its corresponding nuclease, to transform it into a powerful new tool for gene editing1. The article will provide an overview of the origins of CRISPR, the discovery of the CRISPR/Cas9 complex, the potential applications, advantages and limitations of CRISPR/Cas9 and its delivery methods, as well as the prospects of CRISPR/Cas9 technology. plagiarism. Get a tailor-made essay on "Why Violent Video Games Shouldn't Be Banned"? Get an original essayCRISPR refers to many different loci within the bacterial genome. CRISPR loci come from the DNA of viruses that infect the host bacterium. The bacterium is able to incorporate fragments of viral DNA into its genome for the express purpose of producing small segments of RNA known as CRISPR-derived RNA (crRNA)1. The CRISPR-derived RNA then forms a complex with CRISPR-associated proteins (Cas)1 that is able to target and cleave viral DNA. The specific crRNA allows targeting of the viral DNA through the formation of base pairs1, Cas then acts to cleave the viral DNA1 which stops viral replication and provides immunity to the bacterium. There are many CRISPR-associated proteins involved in CRISPR immunity that perform a wide range of functions. In CRISPR gene editing, CRISPR-associated protein 9 is the nuclease responsible for cleavage of the target DNA. CRISPR was originally identified in 1987 by Ishino et al., although only the unique structure was noted at the time. In 2002, the function of CRISPR was identified in a paper by Jansen and Mojica2. A decade later Jinek et al. introduced the CRISPR/Cas9 endonuclease complex. Jinek et al. identified key components of CRISPR and were able to demonstrate the ability to specifically target any DNA sequence for cleavage1. A key part of their discovery was the identification of crRNA and trans-acting antisense RNA (tracrRNA) as the RNAs used in the CRISPR immune response of S. pyogenes1. Jinek et al. were able to design a new “single chimeric guide RNA” (sgRNA) that combined the crRNA and tracrRNA naturally present in S. pyogenes. They also managed to identify Cas9 as the endonuclease acting in the complex capable of creating double-strand breaks in the target viral DNA1. The CRISPR/Cas9-engineered sgRNA is capable of targeting any 20-nucleotide DNA sequence, as long as the target DNA contains another key component of the machinery. In order for Cas9 to become activated and perform target DNA cleavage, Jinek et al. identified the requirement for the presence of a proto-spacer adjacent motif (PAM) immediately following the 20 nucleotide target sequence1. PAM is a three-nucleotide sequence consisting of any nucleotide followed by two glycine nucleotides. Using an engineered CRISPR/Cas9 complex, researchers are able to exploit biological DNA repair mechanisms to introduce or remove genes once a double-strand break (DSB) is achieved with Cas91. The two mechanisms exploited by CRISPR/Cas9-mediated gene editing are non-homologous end joining or (NHEJ) and homology-directed repair (HDR)3. In NHEJ, DSBs are repaired without the aid of a homologous template strand. The two strands of DNA come.