The CRISPR-Cas System

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 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

Ryland F. Young III’s article, “Secret Weapon,” begins by explaining the efficiency of CRISPR defense. Bacteria’s are infected by bacteriophages 1030 times a day. Bacteriophages mutate and undergo an unfathomable amount of genomic recombination that makes it difficult for bacteria to defend themselves from these viruses. Scientist discovered a new bacteria defense systems called CRISPR. This process in combination restriction-modification, restriction enzymes and apoptosis allows bacteria to protect themselves from invading viruses. CRISPR defense is effective because it forced bacteriophages to undergo intense recombination shuffling down to a scale of the size of CRISPR spaces, instead of just mutating. Though CRISPR defense is an effective

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.

Clustered regularly interspaced short palindromic repeats or CRISPR is an efficient and reliable ways to make precise, targeted changes to the genome of living cells. It is a naturally occurring defence mechanism of bacteria. The first part of the defense system is CRISPR and it just remembers parts of viruses DNA so it can recognize and defend against the virus. The second part of the defense mechanism is a set of enzymes called Cas9 or CRISPR associated proteins. Cas9 precisely cuts DNA. The CRISPR/Cas9 system was found in streptococcus pyogenes or better know as the bacteria that causes strep throat. All of this is just shortened to CRISPR. Basicly Cas9 cuts DNA and CRISPR tells it where to cut. All biologists have to do give the Cas9

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

(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

In the past decade, our world has witnessed and produced some of the most unfathomable technological advances that seem utterly unimaginable and futuristic but is now being developed, unfolding right before our very eyes. These advances include: TV screens as thin as a DVD, high-speed internet, smartphones, and even self-driving cars. Today, we have an advancement that could potentially alter the future of our world, indefinitely. “CRISPR-Cas9”, the remarkable technological advancement that is capable of altering a human’s DNA. Scientists must take extreme caution to the ever growing technology in order for ourselves to be the beneficiary of these advancements.

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.

First discovered in 1987, the CRISPR-Cas system is a prokaryotic adaptive immunity defense system that confers resistance against invading nucleic acids, either viral or plasmid. In other words, if an intracellular parasite (such as a phage or a plasmid) invades the bacterial or achaeal host cell, the host cell is capable of storing short fragments of the foreign nucleic acid originating from the parasite within its own genome (Wiedenheft et al. 2012). The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR associated (Cas) genes are found in approximately 40% of bacterial genomes and in nearly 90% of sequenced archaeal genomes (Zhang and al. 2014).

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

This easy-to-use technique will facilitate understanding genome functions and their relationships. It has sparked a revolution in genome engineering field since 2012. Below we review the history of CRISPR-Cas9 system development, reveal its underlying molecular mechanism and discuss its applications, challenges and future avenues of this novel

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