About this sample
About this sample
2 pages /
2 pages /
CRISPR/Cas9 is a method of gene editing that relies on an endonuclease and eukaryotic DNA repair mechanisms. Shorthand for clustered regularly interspaced short palindromic repeats1, CRISPR originally existed as a natural immunity of bacteria against viral infection. Since the discovery of CRISPR researchers have been able to identify, isolate, and modify the mechanism of CRISPR and its corresponding nuclease, to make it into a powerful new tool for gene editing1. The paper will offer 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, and the outlook of CRISPR/Cas9 technology.
CRISPR refers to many different loci within the genome of bacteria. CRISPR loci come from the DNA of viruses that infect the host bacterium. The bacterium 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. 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 for targeting of viral DNA through the formation of base pairs1, Cas then acts to cleave the viral DNA1 which disrupts viral replication and provides immunity to the bacterium. There are many CRISPR-associated proteins involved in CRISPR immunity that perform a wide array of functions. In CRISPR gene editing CRISPR-associated protein 9 is the nuclease responsible for cleavage of target DNA.
CRISPR was originally identified in 1987 by Ishino et al., although at the time it was only the unique structure that was noted. 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 the key components of CRISPR and were able to demonstrate the ability to specifically target any sequence of DNA 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 engineer a new “single chimeric guide RNA” (sgRNA) that combined the crRNA and tracrRNA naturally found in S. pyogenes. They were also to able to identify Cas9 as the acting endonuclease in the complex capable of creating double stranded breaks in target viral DNA1. The engineered sgRNA in CRISPR/Cas9 is able to target any 20-nucleotide sequence of DNA provided that the target DNA contains another key component of the mechanism. In order for Cas9 to activate and perform cleavage of the target DNA, Jinek et al. identified the need for the presence of a proto-spacer adjacent motif (PAM) immediately following the 20-nucleotide target sequence1. The 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-stranded break (DSB) has been 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 are re-ligated at the point of the break with a chance for insertions or deletions during the process. Though NHEJ is capable of inserting exogenous DNA during the repair it will more typically result in deletions of endogenous DNA1. This means that NHEJ is better suited for producing knock-out organisms. The other mechanism, HDR, requires a homologous template strand of DNA from which a copy strand is made to repair the DSB. HDR can be exploited by introducing and engineered homologous template strand that contains a mutated form of the gene being repaired. Due to the nature of HDR, it is better suited to creating knock-in organisms1.
CRISPR/Cas9 is an advancement over current technologies because it offers greater ease, lower cost, and greater effectiveness than current methods1. The applications of CRISPR/Cas9 are wide-ranging and include but are not limited to the creation of animal models, gene therapy, and CRISPR gene interference/activation2. Drawbacks to the use of CRISPR/Cas9 involve the potential for off-target effects that come from slight differences in PAM sequences, or accidentally introduced insertions or deletions during repair of the DSB. Another drawback is the requirement of PAMs. Although any 20-nucleotide sequence can be targeted, cleavage will only occur in the presence of a PAM sequence2.
The outlook for CRISPR/Cas9 is astounding, the number of research papers on CRISPR/Cas9 since its discovery has climbed to nearly 800 published works, the number of CRISPR related patents filed in 2014 was greater than 150, and perhaps the most promising indicator, the amount of funding for CRISPR research has more than quadrupled in 2014 from the previous year4. An article published in Forbes magazine has shown that number has at least doubled in 20155. The amount of funding pouring in to CRISPR research is largely due to the excitement over its potential use in gene therapy. Preliminary studies have shown potential hurdles over delivery methods of CRISPR/Cas9. One team of researchers attempted in vivo gene therapy on a group of mice and had a dismal 0.4% success rate, this was mainly due to the delivery method required. In order to deliver treatment to the livers of the mice, the researchers had to pump in high volumes of liquid into the circulatory system of the mice4. This delivery method would not scale up well for larger animals. Nevertheless, CRISPR/Cas9 remains extremely promising in the world of gene therapy especially given that its greatest setback is the lack of effective delivery methods.
Browse our vast selection of original essay samples, each expertly formatted and styled
Where do you want us to send this sample?
Be careful. This essay is not unique
This essay was donated by a student and is likely to have been used and submitted before
Download this Sample
Free samples may contain mistakes and not unique parts
Sorry, we could not paraphrase this essay. Our professional writers can rewrite it and get you a unique paper.
Please check your inbox.
We can write you a custom essay that will follow your exact instructions and meet the deadlines. Let's fix your grades together!