CRISPR/Cas9 is a gene editing method that relies on endonuclease and eukaryotic DNA repair mechanisms. Short for clustered 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 mechanism and its corresponding nuclease, to make it a powerful new gene editing tool1. The article will provide an overview of the origins of CRISPR, the discovery of the CRISPR/Cas9 complex, potential applications, advantages and limitations of CRISPR/Cas9 and its implementation methods, and the outlook for CRISPR technology /Case9.Say no to plagiarism. Get a tailor-made essay on “Why Violent Video Games Should Not Be Banned”? Get the original essayCRISPR refers to many different loci in the genome of bacteria. CRISPR loci come from the DNA of viruses that infect the host bacteria. The bacteria is able to incorporate snippets of viral DNA into its own 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 capable of targeting and cleaving viral DNA. The specific crRNA helps target viral DNA through the formation of base pairs1, Cas then acts to cleave the viral DNA1, which disrupts viral replication and confers immunity to the bacteria. There are many CRISPR-associated proteins involved in CRISPR immunity that serve a wide range of functions. In CRISPR gene editing, CRISPR-associated protein 9 is the nuclease responsible for cleaving target DNA. CRISPR was originally identified in 1987 by Ishino et al., although at the time only the single structure was noted. In 2002, the function of CRISPR was identified in an article 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 were also able to identify Cas9 as the endonuclease acting in the complex capable of creating double-strand breaks in the target viral DNA1. The sgRNA modified in CRISPR/Cas9 is capable of targeting any 20-nucleotide DNA sequence provided that the target DNA contains another key component of the mechanism. For Cas9 to activate and carry out 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 a CRISPR/Cas9 complex, researchers are able to exploit biological DNA repair mechanisms to introduce or delete genes once a double-strand break (DSB) has been made 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 help of a homologous template strand. The two DNA strands are religated at the break point with the possibility of insertions or.