Application Of Crispr Cas System

There are three types of CRISPR systems. The Type II CRISPR system (where the interface is mediated by a single large protein in conjunction with crRNA) is the simplest of the three and it is this one that has been the basis for genome editing.

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeat, referring to the repeating DNA sequences found in the genomes of microorganisms. CRISPR technology allows scientists to make precise changes in genes by splicing and replacing these DNA sequences with new ones. Through these changes, the biology of the cell is altered and possibly affects the health of an organism. The possibilities are endless as this offers opportunities in curing deadly diseases, modifying genes, and changing humanity as we know it. Although bioengineering has been around since the 1960s, CRISPR is significant because of the comparative low costs and the ease of the procedure to

More recently, Kang et al have employed a different approach using a non-viral delivery method for CRISPR-Cas, known as the

Crispr is based on a natural system used by the bacteria to protect themselves from the infection by viruses. When the bacterium detects the presence of a viral DNA, it produces 2 types of short RNA. One of which contains a sequence that matches that of the invading viral DNA. These 2 RNAs form a complex protein called Cas9. Cas9 is a nuclease, a type of enzyme that can cut DNA. When the matching sequence, known as the guide RNA, finds the target within the viral genome, the Cas9 cuts the target DNA, disabling the virus. But this procedure can be used for any DNA sequence at a precise location by changing the guide RNA to match the

CRISPR or Clustered Regularly Interspaced Short Palindromic Repeat, is used to change the DNA. Today, as humans, we have learned how to use CRISPR for what we want it to do. This is a major break in what we know about DNA. For the future we are looking at how we can change DNA and control what the DNA changes to.

This is done by inserting a break in the DNA and tricking a cells natural DNA repair mechanism into introducing the change one wants. In 2012 two studies were conducted by separate groups, to help transform bacterial Crisper-cas9 into a simple, programmable editing tool. The studies concluded that cas9 could cut any region of DNA, by simply changing the sequence of crRNA, which binds to a complementary DNA target. This system was further simplified when Martin Jinek and his colleagues fused crRNA and tracrRNA to create a single guide RNA. Therefore making it so gene editing only requires two components: a guide RNA and the cas9 protein (Aparna Vidyasagar,

This paper is going to focus on the Cas9 system. Cas9 has a general mechanism of how it works and operates. Further studies and modification to the system have introduced more ways for Cas9 to work. The general mechanism is cleaving the DNA intended DNA portion out of the host genome. This was the original defense mechanism used by bacteria against viruses. In this system, small RNA pieces seek out matching DNA sequences tell the Cas9 where to start cutting the DNA that was inserted by the virus. After the DNA had been cut it was put back together by repairing mechanisms called non homologous end joining (NHEJ) which will be explained later in the paper. One variation of this mechanism is called “nicking”. This modified the Cas9 nuclease to only cut one strand of DNA at a time. The benefit of nicking is that you are able to get a more specific cut of the DNA strand. Two mutations in the nuclease that are relevant for this mutation are. Not only does CRISPR Cas 9 cut out genes it has also been modified for turning on fluorescence. Another function Cas9 can do is break open DNA to make it possible for the insertion of more genes. All of these

The CRISPR-Cas9 complex is derived from the immune system of bacterial cells and also contains repurposed exogenous RNA’s responsible for editing the human genome.1 To edit a gene within the genome, researchers add a CRISPR RNA (crRNA), which is complementary to the DNA code of interest and is responsible for binding. The crRNA is engineered to be extremely specific to the code of interest. A trans-activating CRISPR RNA (tracrRNA) is also necessary for guiding all the pieces of the complex together to carry out the function of editing. The repurposed CRISPR-Cas9 complex contains two nucleases, RuvC and HNH, which perform noncomplementary strand cleavage and complementary

Clustered regulatory interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) are an immune response evolved by bacteria and archea as an adaptive defense mechanism to invading DNA. (4) The CRISPR Cas9 system relies on the uptake of invading DNA fragments that are then inserted into CRISPR loci. (4) In the CRISPR loci, repeats are separated by nucleotide spacers which match and or composed of invading DNA.(4) New spacer DNA is incorporated by Cas1 and Cas2.(4) The CRISPR spacer loci then transcribe into short CRISPR RNAs (crRNA) which anneal to foreign nucleic acids in conjunction with complementary binding trans-activating cr RNA(tracrRNA) to form a duplex which is then cleaved to provide a guiding RNA cr/tracr RNA hybrid.(4) the RNA hybrid acts as a guiding mechanism for Cas9 by complementary binding to the invading nucleotides.(4) Cas9 is an endonuclease that can cause a double stranded cleave in DNA(4) Cas9 guided with sgRNA then cleaves the foreign DNA resulting in double stranded breaks effectively disrupting and thereby removing a gene.(1)(2)(3)(4) After a ds break occurs cellular machinery attempts to fix the break with non homologous end joining in which cellular systems effectively sutures the broken ends of the DNA by recombining the remaining ends of DNA to once again produce a continuous strand.(4) This

The CRISPR system uses two types of molecules, one of which is enzymes. One of the enzymes that can make up CRISPR is called Cas9. Cas9 cuts DNA at a specified location in the genome, determined by the guide RNA. This allows changes to be made in the genome by allowing DNA to be added or removed from

Phages are the most common organisms on Earth (3,7): they infect bacteria by injecting single-stranded DNA into their host cell, thereby altering the genetic code of the prokaryote. Once transcribed and translated, the injected DNA changes the function of the cell, often killing the host cell in the process (3). Over eons, archaea and some bacteria have adapted to such attacks with CRISPR-Cas systems, also known as clustered regularly-interspaced short palindromic repeats. The bacteria incorporate invading viral DNA into their genetic material in between redundant palindromic sequences. The viral DNA is then transcribed into pre-crRNA, where it combines with a Cascade complex. Cascade proteins come from CRISPR-associated (Cas) genes. Such genes code for small enzymes responsible for cleaving. They often join together into larger complexes known as Cascade, or CRISPR-associated complex for antiviral defense (2). The cas proteins use the RNA template as a guide and cut any other genetic material that matches that of the viral-template RNA, preventing the corrupt viral DNA from being transcribed into mRNA. As the number of spacers in the bacterium’s DNA increases, the level of phage resistance increases as well (1). Insofar, understanding of CRISPR and Cas genes is very limited. It is very much a twenty-first century discovery.

Even though CRISPR/Cas9 was just discovered around 4 years ago, the CRISPR/Cas system itself was first observed in bacteria (E.coli). It was what CRISPR was doing in the bacteria that makes it so unique. CRISPR stands for, “clustered regularly interspaced short palindromic repeats”. This means that there are going to be short sequences of DNA bases that will read the same forward and backwards. In-between these short sequences will be spacer DNA. Unlike the palindromic sequences, the spacer DNA will have sequences

Thus, spacers operate as a ‘genetic memory’ of the preceding infections. If an infection caused by the same virus were to occur, the defense system of CRISPR would cut any DNA sequence that matches the spacer sequence, and will consequently protect the bacterium from any viral attacks. If an earlier undetected virus attacks, a new spacer will be created and added to the repeats and spacer chain. The three basic steps that the CRISPR immune system follows to protect bacteria from repeated viral attacks are as follows. Step 1 is adaptation, where the DNA from the attacking virus is managed into short segments, which are injected as new spacers into the CRISPR sequence. Step two is the production of CRISPR RAN, where the CRISPR spacers and repeats that are in the bacterial DNA go through transcription, the method of copying a segment of DNA into RNA. The resultant is a single-chain RNA molecule, which is cut into shorter segments that are called CRISPR RNAs. Step three is targeting, where the CRISPR RNAs will direct the bacterial molecular machinery to terminate the viral

The CRISPR/Cas9 system requires a proto-spacer adjacent motif (PAM) sequence. The PAM is necessary for cleavage of target DNA by Cas9, allowing differentiation between invading viral genome (in this case HIV) and the CRISPR locus in the host genome. One limitation of the CRISPR/Cas9 system to disrupt HIV could be due to low altering specificity, as the target loci is very small, so significant off target cleavage can occur (Wang et al.). CRISPR-Cas9 may create non-specific mutations in areas of the genome with similar homology, outside of the HIV provirus locus, which may cause cellular transformation (Larson et al., Tu et al.). Selection of Cas9 homologs with a longer PAM may decrease the probability of non-specific gene target and Cas9 homologs

This article is centered around the research being conducted on an organic molecule called CRISPR-Cas9, a relatively recent discovery in microbiology that allows scientists to edit the DNA inside of living cells. The author’s thesis is that CRISPR-Cas9 technology has both positive and negative implications, and that several influential establishments are investing in its future.