Essay On Cryptococcosis

For approximately three-thousand years, smallpox has ravaged and plagued the four corners of the globe. In fact, in the 17 th and 18 th centuries, it was claimed to be the most infectious disease in the West, with an astounding 90% mortality rate in America. It wasn't until 1796, with English surgeon Edward Jenner's smallpox vaccination, that the world saw relief from this devastating virus. However, even with this inoculation in use, the world continued to witness death from both the virus and the vaccine. In the year 1966, it was estimated that 10-15 million infected citizens world wide had passed away from smallpox that year alone ( “History” 12). As a result of these devastating numbers, in the following year, 1967, the World Health

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

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.

The title of the most extreme extremophile goes to the chroococcidiopsis. Though its name alone qualifies this cyanobacteria species as extreme, its resume speaks for itself. Able to withstand both extreme high and extreme low temperatures, radiation up to 15 kGy, and UV doses as high as 13 kj/m^2, these cyanobacteria are impressively resilient. Chroococcidiopsis species are adaptable enough to grow as litho-, endo-, chasmoendo-, crypto-, and hypo-liths, survive in several different aquatic environments (fresh, brackish, and salt water) and find home in rocks. Not only that, but chroococcidiopsis is so extreme it is a prime candidate to study in the search for life in the universe.

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.

Figure 2. Schematic illustration of generating Nf1-/- iPS cells from its Nf1+/- counterparts. (A) is the genotype of Nf1+/- iPS cells. (B) and (C) are two kinds of Nf1-/- iPS cells after Crispr/cas9 mediated NF1 genome editing in Nf1+/- iPS cells. In the presence of the repair template, Crispr/cas9 is able to introduce a nonsense mutation (c.94-96>TAA) into the exon 2 of NF1 genome via the homology directed repair (HDR). Without the repair template, random insertions and/or deletions may be introduced by non-homologous end joining (NHEJ). Nf1-/- iPS cells are to be generated by introducing

So, in one study, scientists engineered a modified CRISPR system to work with customized guide RNA (gRNA) in human cells to target genes linked to leukemia, to which they obtained high targeting rates. They proved that this process was not by random chance, but that it was caused by CRISPR components, that the process was sequence-specific and, when they introduced multiple gRNAs, it can affect multiplex editing of target loci. Their results created an RNA-guided editing tool for easy, efficient, and generalized human genome

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

Hypothesis: Ψ-mRNA, synthesized via in vitro transcription, will have enhanced stability and decreased immunogenic properties. Restriction enzymes will be used to clone the NT-3 gene (NTF3) into the pDNA expression vector known as pDNA3.1. DNA polymerase, deoxynucleotide triphosphates (dNTPs), a forward primer containing a T7 promoter, a reverse primer containing a 75 thymine tail, and the pDNA3.1 vector will be combined in a polymerase chain reaction (PCR) tube. Standard PCR techniques

We will perform expression validation of exRNA signatures produced by JEC21, a serotype D strain and analyze whether these exRNA signatures are also present in other serotype D strains of Cn, as well as serotype B and C C. gattii strains using RT-qPCR. We will isolate exRNAs from serotype A Cn strain H99 and Cg strains such as R265 (VGIIa) and R272 (VGIIb). The results will be analyzed to look for common expression profiles of RNA that could serve as molecular signatures produced by Cn and possibly Cg strains that are not produced by mammalian hosts such as humans or mice. This bioinformatics work will be performed under the direction of the in-house bioinformatics specialist, Dr. Chris Taylor. Please see his attached letter of support. Production of the biomarker candidates will be verified by RT-qPCR.

Cas9 has been well studied. It belongs to the class II CRISPR/Cas system which is the main protein involved in small interfering CRISPR RNA (crRNA). CRISPR RNA often leads to the silencing of invader viruses and plasmids [72, 85]. Several in vitro surveys have shown that Cas9 is guided by gRNA composed of chimeric RNA. Chimeric RNA is composed of crRNA and tracrRNA. They are sufficient to

One of the main uses of CRISPR-Cas9 is to generate knock-out cells or animals, however modifications can be made to the Cas9 enzyme to selectively activate or repress target genes or purify specific regions of DNA. Before discussing how CRISPR works, it is important to understand its fundamental parts. There are two components of CRISPR, one being a “guide” RNA (gRNA) and the other being a non-specific CRSIPR-associated endonuclease, which is Cas9. The gRNA is synthetic RNA made up of a “scaffold” sequence for the Cas9-binding and a user-made nucleotide “spacer” sequence. The genomic DNA being addressed can be any 20-nucleotide DNA sequence as long as it meets certain conditions. This includes being unique compared to the rest of the genome and the target is present upstream of a Protospacer Adjacent Motif, or PAM. (“CRISPR/Cas9 Guide”, n.d.).

CRISPR-Cas9 currently stands out as the fastest, most reliable, and least expensive method of manipulating genes. It is also the most versatile and most accurate, thanks to its simplicity. For this reason, it has garnered much attention from both the scientific community and the general public.