BS31006 – Gene Regulation & Expression – Dr. Armel Nicolas
Genome engineering using the CRISPR-Cas9 system
Matei Agavriloaei - 120004682
24/11/2014
ABSTRACT:
Modified nucleases have been used for years in order to achieve successful genome editing and are nowadays an almost universally-used method. RNA-guided nucleases (such as Cas9) with easily changeable characteristics have been generated in great numbers especially since the emergence of clustered regularly interspaced short palindromic repeats (CRISPR). This represents the most modern tool that can be used by scientists for genome editing. This technology can bring a broad range of medical benefits and advancements (1).
CRISPR-Cas systems:
CRISPRs are DNA loci that include
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Figure 1: Overview of the CRISPR-Cas system – it shows the adaptive immunity with the use of viral DNA (3);
Cas genes code for proteins related to CRISPRs. This association is a prokaryotic immune system. CRISPR spacers recognize and cut the foreign DNA material, just as RNAi does in eukaryotic organisms (2).
In 2013, this system started being used (and is still widely used today) for adding and changing sequences of targeted genes (4), process also known as genome editing. The genome can be cut any location by guiding properly RNAs into a cell and also delivering Cas9 protein. In theory, it could be possible to build RNA-guided gene drives with the use of CRISPR to alter the genetic code of entire populations (5).
CRISPR-RNA (crRNA) and Cas proteins come together and form CRISPR-ribonucleic proteins (crRNPs) for appropriate targeting and cleaving of the foreign nucleic acid (3).
Due to the accelerate evolution of the immune system, the CRISPR-Cas systems are highly diverse and have been catalogued into three main types, each of them having a specific Cas protein. Type I and III and related and contain Cas3 nuclease-helicase and Cas10 (a protein with an unknown function) respectively. Type II is phylogenetically different and is represented by the Cas9 nuclease.
Type II has three different subunits (A, B and C) and their crRNPs are all known as Cas9 complexes. They are restricted only to bacteria, not being in present in archaea as well (3).
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 has been garnishing a lot of media attention recently and it is not just popular among the scientific community but also the general public. Several online news outlets and scientific journals have been talking about the significance CRISPR-Cas could have for the field of genetics and science as a whole. I even came across a Youtube video from The Verge, a tech channel that normally does reviews on new smartphones and laptops talking about CRISPR [15]. So why is CRISPR gaining so much attention both from the scientific community and the general public? The answer lies in the potential this technology possesses.
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
The author gives a brief history of past genome editing but thoroughly explains the history and mechanism of the CRISPR technology. She elaborates on how the technology has already been used to cure diseases and speculates on its future uses and regulation.
In “Life the Remix,” Alice Park discusses the impact and influence CRISPR has on science as well as its potential and risks. CRISPR—“clustered regularly interspaced short palindromic repeats”—is a technique to alter DNA, virtually for anything involving DNA. Although there have been attempts to edit DNA, none were as cheap and simple as CRISPR. This technique, which is based on the immune system of a bacetria, revolutionizes genetics after the subsequent discoveries of the molecular scissors enzyme: Cas9 and a method to efficiently and accurately edit human DNA using CRISPR, explains Park.
More recently, Kang et al have employed a different approach using a non-viral delivery method for CRISPR-Cas, known as the
Every few years, advancements in technology alter the way scientists do their work. Recently CRISPR-Cas9, a RNA useful for working organisms in the animal kingdom has proven itself beneficial on a gene-editing platform. After performing many abortive attempts to manipulate gene function, including homologous recombination and RNA interference, scientists have finally had a breakthrough with CRISPR-Cas9.
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 is versatile in that any target sequence can be modified by simply altering the gRNA sequence. In addition, multiple genes can be edited at the same time with great specificity (Cong 88-89). The convenience and accessibility of CRISPR resourced have also allowed thousands of laboratories worldwide to study CRISPR in different ways, which has broadened the horizons of its biomedical and clinical implications (Collins et al 259). Overall, the ease and simplicity of CRISPR technology has allowed for a rapid increase in the understanding of genome editing, which will allow CRISPR to revolutionize how certain conditions will be treated.
The ability to engineer biological systems and organisms has an enormous potential for applications across basic science, medicine and biotechnology. Genome editing is a group of technologies that allow scientists the ability to change an organism’s DNA, which can provide better outcomes for health and disease control compared to natural immunity and mutations. Genome editing (gene editing) allows genetic material to be added, removed or altered at particular locations in the genome. A number of gene editing technologies have emerged in recent years with one of the most versatile and precise methods of genetic manipulation being Crispr-Cas9 (Steve scott,2016). The term Crispr-cas9, (clustered regular interspaced short palindromic repeats)
This article is about CRISPR its function and the concerns. Ledford talks about the benefits and also improvements that the first CRISPR needs. She shows how this technology not only could benefits humans but animals and plants as well. This article is useful in that it informs the public about the progress of CRISPR.
The Cas9 follows the guide RNA to the same location in the DNA sequence and makes a cut across both strands of the DNA. At this stage the cell recognises that the DNA is damaged and tries to repair it. Scientists can use the DNA repair machinery to introduce changes to one or more genes in the genome of a cell of interest. According to a team of scientists working on CRISPR, “The guide RNA is designed to find and bind to a specific sequence in the DNA. The guide RNA has RNA bases that are complementary to those of the target DNA sequence in the genome. This means that, at least in theory, the guide RNA will only bind to the target sequence and no other regions of the genome.” The CRISPR-Cas9 has a gene editing
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