Nucleaser Research Paper

The Cas9 protein is an enzyme that effectively acts as a pair of molecular ‘scissors’, cutting

Mullis came to light. This technology seemed to to hold a promise that it would end human suffering, that it would be the road to a perfect world, where diseases were no longer a threat and pesticides would become an archaic method of the past. This new technology was called PCR, and it was the earliest form of gene editing. Fast forward to today, where another great leap in the science of gene editing has just occurred - one that might be exactly what everyone thought PCR would turn into. This leap has been dubbed CRISPR, and its capabilities make PCR look like, well, nothing. CRISPR uses a device originally found in bacteria called CAS-9 to precisely snip a targeted area of an organism's genome and replace it with the correct gene. CRISPR is by all accounts an amazing technology, but there are some who think it should not be used. CRISPR has

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-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

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.

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

(2012) introduced a new genetic technique that was derived from the defense mechanisms of bacteria. Some bacteria use a CRISPR-Cas system to defend against foreign viral and plasmid genetic material. Once foreign targets enter the system, the bacteria will integrate its CRISPR array to parts of the nucleotide sequences on the invading sequence. The bacteria will then produce a precursor CRISPR-RNA that complements the invading sequence, and is used to find all foreign sequences that match it. These precursor RNAs will work with Cas proteins to cleave the foreign sequence, thus effectively silencing it. There are multiple types of CRISPR-Cas systems that bacteria use. Type 2 systems, paired with Cas-9, use another RNA sequence, tracrRNA (trRNA), as a complement to precursor CRISPR-RNA. These systems used both trRNA and precursor CRISPR-RNA to induce a double stranded cleave. After this discovery, a Cas9 protein was purified and tested to see if it would be able to cleave DNA. It was discovered that if both a trRNA and a precursor CRISPR RNA were present with complementary sequences to a sequence in a DNA strand, the result would be a double strand cleave in the DNA. Cas9 also contains two domains, each of which only cleave either the complementary or the non-complementary strand of the target DNA. After looking at both the trRNA and the precursor CRISPR RNA, researchers theorized that they could engineer a chimera RNA that combined certain sequences of both

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

Then, like in the targeting step of the bacterial system, this ‘guide RNA’ shuttles molecular machinery to the intended DNA target. Once localized to the DNA region of interest, the molecular machinery can silence a gene or even change the sequence

The eleven double-stranded RNA segments remain within the protection of the two protein shells. The RNA dependent RNA polymerase of the virus creates mRNA transcripts of the double-stranded viral genome. By remaining in the core, the viral RNA evades innate host immune responses known as RNA interference that are triggered by the presence of double-stranded RNA.

The Cas9 System and a small guide RNA molecule. The Cas9 is an enzyme that “snips through DNA like a pair of molecule scissors”(Zagorulya). The second component is a RNA molecule that acts as a guide to direct the Cas9 to the targeted sequence of DNA in the genome. DNA repair mechanisms in the cell can silence the gene if cut. When adding a new DNA fragment to the Cas9-gRNA complex, repair machinery repair DNA by adding the new DNA where the enzyme cut, through the DNA. With this, a mutation can be introduced into the gene or a new gene can be introduced into the cell DNA. Scientists are able to manipulate DNA in numerous ways using the system and study the function of health and mutated forms of specific genes. The flexibility of this system allows scientists to study any gene despite its location or composition of DNA. Flexibility is due to the RNAs design as it is created to target any site of the

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.

In this TED talk, biologist and geneticist, Jennifer Douda, presents a way to edit our DNA with the use of CRISPR technology. This technology allows scientists to change the DNA within our cells that could ultimately allow scientists to cure various genetic disease such as sickle cell anemia, Huntington’s disease and cystic fibrosis etc. The way this technology works is that the Cas9 protein seeks foreign DNA, and precisely cuts that DNA out and eventually degrades it. After the mutated DNA is removed from the double helix, the cells detect the broken DNA double helix, and repairs it by pasting its ends together via a tiny change within the sequence of the DNA, or cells may simply introduce a new sequence of DNA at the site of the break,

The protective capsid helps the virus escape detection and destruction during the invasion of the host. When the virus reaches the target cell, biochemical reactions between the capsid and cell wall allow the virus to latch on and inject its genome into the cell’s interior. Once inside, the viral genetic material insinuates itself into the host’s DNA or RNA. In an efficient feat of natural bioengineering, the host cell’s genetic machinery now does the rest of the work for the virus. The cell, which had already been making copies of its own genome, now also replicates that of the virus. Coded within the viral material is the blueprint for making more copies of the viral genome. Further instructions command the production of capsids and directions for assembly of new viruses. After the host cell becomes engorged with viruses, it explodes, sending the new