CRISPR Pathway Analysis

There are many highly diverse Cas (CRISPR associated) proteins but only one that is mainly used, which is Cas9.1 Which Cas9 derivative is found on the gRNA that contains plasmid, which are associated with CRISPRs type II in the bacterial immune system. Cas9 is an important asset to a type II CRISPR system mechanism that is required for gene silencing. Cas9 protein engages in the processing of crRNA and is also responsible for eliminating the target DNA in the type II CRISPR system.1 Cas9 function as an RNA guide for the DNA endonuclease enzyme that is involved with CRISPR adaptive immune system.

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 is not an artificial construction; bacteria use it as a form of adaptive anti-viral immunity. When infected with a pathogen, bacteria that has this system retain a portion of the infecting virus in their chromosomal DNA. The cell then transcribes those sequences (called CRISPRs, or “clustered regularly interspaced short palindromic repeats”) and processes them into short RNAs called crRNAs. The crRNA guides the Cas9 nuclease to its target (normally, an invading viral nucleic acid) and cuts the foreign nucleic acid, rendering the virus useless in the process.

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

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

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.

CRISPR immunity involves the three following phases: acquisition, expression and interference. During the acquisition stage, new spacer sequences are incorporated into the CRISPR locus. During the expression stage, the CRISPR array is transcribed into a premature crRNA (pre-crRNA) and then matures into the CRISPR RNA (crRNA). In the last stage, the foreign DNA is destroyed using the processed crRNA in some form of effector complex containing Cas proteins (Weidenheft et al., 2012) (Figure 3).

The cheapest method, CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats allows for the genome to be cut and spliced back together using the Cas9 nuclease. These changes are permanent, and will be passed down to future generations. However, there are concerns that this method of gene editing could slow down and eventually stop human evolution, and CRISPR editing is not as precise as other methods. The Cas9 nuclease has no problem with cutting out parts of DNA, but it often struggles with filling the gaps it creates. The replacing DNA strands are sometimes guided into the wrong place, resulting in unwanted, unneeded, and permanent changes to the genome that can do more harm than

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

Relatively recently, CRISPR-Cas9 has emerged as a revolutionary tool for gene editing. Its programming simplicity, ease of use, and inexpensiveness distinguishes it from other methods that perform the same task. There are specific loci in the genome of bacteria and many archaea containing repetitive sequences “interspaced” by non-repetitive sequences of similar length (Jansen, Embden, Gaastra, & Schouls, 2002), leading to the term “Clustered Regularly Interspaced Short Palindromic Repeats.” The CRISPR-Cas system, as part of the bacteria’s adaptive immune system, are used as a defense mechanism against bacteriophages and plasmids, and a portion of the DNA of the virus is integrated into these CRISPR loci. The RNA transcribed from the loci can

CRISPR-cas9 allows researchers to edit part of the genome by inserting, deleting, or altering sections of the DNA sequence. The CRISPR-cas9 system consists of two key molecules: Cas9 and single-guide RNA (sgRNA). The enzyme Cas9 is ‘guided’ by sgRNA to the DNA sequences where it cuts the two strands of DNA at the specific location in the genome. Endogenous DNA repair mechanisms try to repair the breaks in both strands which then leads to indels that can cause mutation in the target gene. This process is shown in Figure 1.1. To determine whether CRISPR–Cas9 can be used to understand gene function in human preimplantation development, the POU5F1 gene that encodes the development regulator OCT4 was selected as the target. It was predicted that

Researchers have moved one step closer to developing a cure for AIDS, the killer which claimed 39 million lives since its discovery in the 1980s. Using the relatively new CRISPR/Cas9 technology, scientists at the Lewis Katz School of Medicine at Temple University have eliminated HIV-1 genomes from human T-cells. CRISPR/Cas9 is a powerful biological tool used for genome editing and altering gene expressions. Applications of CRISPR/Cas9 range widely from modifying yeast to produce biofuels, to inactivating genes in human cell lines. Kaminski et al. (2016) believe that CRISPR/Cas9 is the key to curing AIDS because it holds more potential compared to the current method, antiretroviral therapy (ART). ART uses multiple medications to combat against

Since CRISPR-Cas give scientist the ability to edit genomes with precision and efficiency, the applications of using CRISPR-Cas in space are endless. Scientist could manipulate the DNA of humans so that mutations provide astronauts with denser bones and larger amounts of muscle mass. Researchers can also use CRISPR-Cas to edit the genome of astronauts so that they resemble the mutations found in “Superhumans.” These mutations would make astronauts less vulnerable to bone loss and many other effects experiences in space travel. In addition, scientist could splice human DNA with other organisms through a process called transgenesis that may possibly make astronauts more resistant to radiation and its effects on the body outside of the earth’s magnetosphere.

Additionally, CRISPR-Cas mediated immunity develops through the intake of DNA from genetic elements such as plasmids and phages followed by its incorporation into the CRISPR loci. It is within the CRISPR loci where the small interfering RNAs are processed and transcribed that aid the nucleases for precise cleavage of corresponding sequences. Interestingly enough, recognizing the genetic sequences in CRISPR loci give a clear insight into vaccinations events that the prokaryote would’ve gone through, and can give some perception in the environmental changes occurring over a period of time. Therefore, cas endonucleases, which can be reengineered by small guide RNAs, have proven to demonstrate remarkable development and flexibility for genome editing and can be repurposed for a number of DNA purposing applications. The main scope of the report will focus on the structure and function of the CRISPR-CAS system, the organisation of the CRISPR loci and its significance, as well as contrasting some differences between prokaryotes in CRISPR guide RNA (crRNA) design.