The Catalytic Duty Of The Crispr Cas Complex

Once the complex was bound to the DNA, a cut would be made to eliminate and destroy the invaders. 83% of archaeal genomes and 45% of bacterial genomes (Shabbir, M. et al, 2016) were shown to be able to successfully utilize the CRISPR Cas9 system. These are very promising statistics, so it is no wonder that there has been such an advancement in the past few years to bring this technology to eukaryotic cells, mammalian cells and eventually human cells.

Since the 1980’s, scientists observed a unnatural patterns in some bacterial genomes. One DNA sequence would be repeated over and over again, with original sequences in between the repeats. They called this odd function “clustered regularly interspaced short palindromic repeats,” or CRISPR. This was confusing to all until scientists realized the unique sequences in between the repeats matched the DNA of viruses, specifically viruses that target certain bacteria. It turns out CRISPR is an important part in the bacteria’s immune system, which keeps harmful viruses around so it is recognizable and can defend against those viruses next time they attack for an example; kind of like a book one would keep around to look back on for help even though one does not need the book at the time. The second part of the defense mechanism is a set of enzymes called Cas or, CRISPR-associated proteins, which can precisely locate DNA and remove all of the invading viruses. Conveniently, the genes that encode for Cas are always sitting somewhere near the CRISPR sequences kind

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

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.

Genetic diseases and illnesses have been of much concern for many years, leaving many deceased or with a poor quality of life. Due to the implication of modern medicine and other techniques used for treatments, mortality rates have decreased and the average life expectancy has increased. Unfortunately, every individual responds differently to the type of treatment they need, which is why the implication of personalized medicine is forthcoming. A certain technique that has been distinguished and commended by researchers today is known as clustered regulatory interspaced short palindromic repeats, or CRISPR. CRISPR is associated with Cas9, and it is a popular genome editing technique which can be programmed to target specific areas of DNA and

Even though it was discovered in 1987 by a research team at Osaka University, the true power of CRISPR, which functions as an adaptive bacteria immune defense, was not realized until the completion of the Human Genome Project in the early 2000’s (Carroll and Zhou 63-64). There are only two components of CRISPR, a guide RNA sequence (gRNA) and a Cas9 endonuclease protein. When the gRNA binds to its matching template DNA sequence, the Cas9 protein cuts the template DNA sequence resulting in the inactivation of that specific gene (Hille and Charpentier 3). In addition, a mutant gene can be replaced by adding another piece of DNA with the desired sequence, which will bind to the cut DNA template strand and become incorporated into the host

CRISPR-Cas9, a genome editing instrument, moves to change the field of biology forever. CRISPR was first observed as an innate defense mechanism used by bacteria. After years of development, scientists have been able to construct their own RNA that guides the CRISPR-Cas9. This allows them to control the behavior of the CRISPR-Cas9. What this could mean for the future is overwhelming.

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 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR Associated System (CAS) is an advancement in technology that transformed a previous protein-based targeting (TALEN and Zinc Finger) process used to target and splice genes or DNA sequences. CRISPR-Cas9 was discovered in 2013 by Feng Zhang from the Broad Institute and MIT. CRISPR-Cas9 targets specific base pairs using small RNA that can be easily swapped for many different RNA targeting sites. This allowed CRISPR-Cas9 to surpass the previously used splicing methods such as, Transcription Activator-Like Effector (TALE) and zinc fingers. TALEs and zinc fingers are banded to DNA through a direct protein-DNA interaction. This was a long and tedious process that required proteins to be redesigned for each new target DNA site. CRISPR-Cas9 made the process of genome editing more efficient, effective, and precise by using RNA and proteins (“CRISPR”). CRISPR-Cas9 is a natural system that helps bacteria defend against attacking viruses known as bacteriophages. This process begins with the “CRISPR sequences [bookending] short stretches of DNA that bacteria have copied from invading phages, preserving a memory of the viruses that have attacked them in the past” (“CRISPR”). These sequences are then transcribed into short RNAs that guide the Cas proteins to match the viral sequences. The Cas proteins destroy the matching viral DNA by cutting it from the strand.

CRISPR Cas9 is a gene editing tool that can be used to edit, delete, and change parts of the genome. What makes CRISPR Cas9 different from other gene editing techniques such as Zinc Finger Nuclease (ZFN) or Transcription Activator-Like Effector Nucleases (TALEN) is its targeting efficiency ability. For example, ZFNs and TALENs could only reach targeting efficiencies from 1% to 50% in human cells. On the other hand, CRISPR Cas9 had a much higher rate at greater than 70% in Zebrafish and plants. (Reis, 2014). CRISPR Cas9 is still being heavily researched by scientists today, because although many advancements have been made since its discovery in 1993, it is far from ready to be used commonly. Successful studies and experiments with this gene

Gene editing is the technique using to edit specific parts a genetic sequence to express or suppress a characteristic in an organism. In 2002, Ruud Jansen performed an “in silico analysis” to study a family of repetitive DNA sequences in only prokaryotes (Jansen 2002). In silico analysis is a type of testing done by using a computer simulation (“In Silico”). The researchers called the families clustered regularly interspaced short palindromic repeats or CRISPR (Jansen). They found that cas genes were always adjacent to CRISPR loci which indicated a functional relationship between the two.

The endonuclease Cas9 is the core multifunctional protein of the CRISPR-Cas9 system. Two nuclease domains: HNH (39) and RuvC-like (40), within the Streptococcus pyogenes Cas9 (SpCas9) protein conduct the function of cleaving DNA strands. Studies have confirmed that the tracrRNA: crRNA duplex structure will lead to the sequence-specific DNA cleavage (41). PAM short sequence facilitates crRNA to recognize the targeted DNA sequence (42). Cas9 protein cuts the specific DNA strand that is complementary to the 20bp crRNA sequence via HNH domain; on the other hand, the SpCas9 RuvC-like domain cleaves the opposite targeted DNA strand (42) (Fig. 4). Mutating either of these two nucleases domains of SpCas9 will introduce a single-stranded DNA nick, while mutating both HNH and RuvC-like domains will transform SpCas9 into a RNA-guided DNA binding protein (42). X-ray crystallography and has confirm that endonuclease Cas9 undergoes a series of conformational rearrangements when it binds to the guide RNA and target DNA (55).

This RNA then joins with the CRISPR associated (Cas) proteins. In this way the RNA guides and finds its matching viral DNA (by complementarity) and the Cas protein chops it up and destroys the virus (Fig 1). Discovering the basic function and mechanism of CRISPR encouraged a variety of groups to explore how they could harness it and apply it to systems of their choice. This was the new big hit, the CRISPR revolution was born (Table 1). The essential components required to reconstruct the CRISPR/Cas system were identified (Deltcheva et al., 2011), it was also shown possible to transfer and express those component between organisms (Sapranauskas et al., 2011) : the power of CRISPR was becoming apparent (Jinek et al., 2012). In 2013 this power was applied to genome editing made possible for the very first time in eukaryotic and mammalian cells (Mali et al., 2013) (Cong et al., 2013).

The process in which the CRISPR pathway works contains a few steps that, after assembly of the RNA and nuclease complex, will occur naturally in the cell. From this point forward, the RNA sequence that is engineered to bind specifically to a target sequence will be referred to as guide RNA (gRNA). The assembly of the gRNA and Cas9 endonuclease complex binds to the target gene sequence and cleaves at a point just downstream of the protospacer adjacent motif (PAM; Figure 1; Chang et al., 2013) The PAM is simply a sequence recognized by the Cas9 as ‘non-self’ to allow for cleavage by the endonuclease (Chang et al., 2013). If this were not present, the bacteriophage sequences incorporated into the host bacterial DNA initially, would be targeted by the bacteria’s CRISPR system for cleavage.

CRISPR stands for 'clustered regularly interspaced short palindromic repeats'. These are short DNA sequences that are found in bacteria. This is used to make RNA that along with a protein called Cas9, to make cuts in the invading viral DNA. This mechanism is used by bacteria to protect and defend against invading viruses.