BICD1 Case Study

Hereditary spastic paraplegia (HSP) is a heterogeneous class of diseases marked by lower limb weakness and spasticity due to progressive degeneration of axons in the corticospinal tract. HSP has been linked to mutations in the neuronal motor protein, kinesin Kif5A. The function of this protein is to transport cellular cargo from cell bodies to synaptic terminals. We hypothesize that the disease is caused by ineffective transport of cellular cargo. Fifty percent of the known disease-causing mutations are in the region of Kif5A that interacts with microtubules. We have previously shown that disease-causing missense mutations in the motor domain of Kif5A cause a reduction in kinesin motility rate, as well as reduced microtubule binding affinity.

The process for generating amino acid coding changes at the DNA level is called directed mutagenesis. Due to the lack of well characterized three

A study was conducted that questioned the role of the organization of copy number variants and wondered what type of duplications they are. It was known that that copy number variation plays a big role in many human diseases but at the time large scale studies of these duplications had not been done. They decided to sequence 130 breakpoints from 112 individuals that contained 119 known CNVs by doing whole genome sequencing as well as next generation sequencing. They found that tandem duplications comprised 83% of the CNVs while 8.4% were triplications, 4.2% were adjacent duplications, 2.5% were insertional translocations, and 1.7% were other complex rearrangements (Newman et al., 2015). The copy number variants were predominantly tandem duplications which made it the most common type of copy number variant in the human genome according to the results of the study on this

A simple but effective method is correctly designing gRNAs. Ideally, the target sequence must be a unique and nearly perfect homology to the gRNA with as little to no homology at other points within the gene sequence. Another potential strategy involves reducing the concentration of gRNA and Cas9 that are expressed in cells. However, this strategy is considered inconclusive due to inconsistent reporting. Some cell lines showed a large decrease in rates of off-target binding while others showed no effect. An increasingly promising method for improving specificity involves using a mutated nickase version of the Cas9 enzyme. This modified version contains only one catatylic domain and is used as a single nickase system capable of cutting a single strand of DNA. This results in a “nick” or single stand break rather then a DSB and is repaired by HDR. To further reduce the risk of off target binding, a double or dual nickase CRISPR system can be implemented. This system uses two nickases which bind to opposite strands of the target gene, generating a DSB. This system closely resembles the Fokl enzyme activation used in TALEN, in that off target effects are reduced because the probability of two off target nicks within the same region is very unlikely. When the DSB is made, activation of NHEJ is initiated; to further increase specificity and reduce off-target effects, the HDR mediated editing is advised for both nickase

Firstly, shot-gun sequencing requires a large portion of template DNA for each read, and subsequently, numerous strands of template DNA are required for each read since a strand that terminates on each base is required for the construction of a complete sequence. However, a sequence can be achieved through a single strand using next-generation sequencing. Also, next-generation sequencing is a faster process than shot-gun sequencing since the chemical reactions taking place in next-generation sequencing can be merged with signal detection, which cannot be done with shot-gun sequencing, and next-generation sequencing allows more DNA to be read on a single run than compared to shot-gun sequencing. Additionally, next-generation sequencing results in reduced costs since not as much human labor and reagents are required as those needed for shot-gun sequencing. Furthermore, next-generation sequencing is more accurate than shot-gun sequencing as next-generation sequencing involves numerous repeats due to the need for each read to be amplified before sequencing and its dependence on numerous short overlapping reads, in order for multiple sequences of DNA and RNA to be achieved. Also, due to its cheap costs, it would be more economical to perform multiple repeats rather than shot-gun sequencing, whose costs are exceedingly greater. Therefore, due to

Genes are the foundation of life. Biologists throughout history have studied genes with the goal of knowing how they work, what functions specific genes have, and what they can do with this knowledge to further science and society. Techniques involving genes have grown to the level where we can target specific genes to edit them and even alter their function. Some earlier techniques of this include meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases, but each of these techniques has its disadvantages (Sander and Joung, 2014). Recently in the world of genetics, a new and exciting method to target specific genes easily and with high precision has emerged. Clustered regularly interspaced short palindromic

The initial Sanger sequencing method has been administered to several significant improvements and developed remarkably over three decades. Even though cloning of DNA fragments into plasmid vector was required in Sanger sequencing, the application of polymerase chain reaction (PCR) (Saiki et al., 1988) for the amplification of specific DNA fragment in vitro has extensively

The data collected from ExAC consisted of targeted exome sequencing using XHMM. XHMM used an algorithm to filter out individuals who were not likely to have the target genes for the CNV calling. In total, they attempted CNV calling for 60,642 of the 60,706 ExAC samples, with the remainder either having failed calling for low overall read depth or not included because of technical issues. Genes were filtered out based on the type of chromosome, unusually low or high mean coverage, multiallelic genes, and CNVs with a frequency greater than 0.5%. The authors then presented a histogram of the number of copy number variations per individual, a 2D-density plot of copy number variation number and length of CNVs, and a density plot of the amount of copy number variation per individual. They also included a table of the number of total genes affected and mean number of gene-level CNVs per individual, as well as two bar charts representing the data in the table. The purpose of the statistical analysis and modeling included in the paper was to show a data-driven way for approximating the likely deleteriousness of genic CNVs.

A new tool based on a bacterial CRISPR-related protein-9 nuclease (Cas9) from Streptococcus pyogenes is an effective and reliable method for making precise, targeted changes in the genome of living cells. The functions of the CRISPR (Short Palindromic Repeats Clustering Regular Intermediate Repeats) and CRISPR-associated (Cas) genes in adaptive immunity in selected bacteria and archetypes allow the organisms to respond to and remove invasive genetic material.

The HGP employed a two-phase approach to uncover the human genome sequence. The first phase used DNA shotgun

Site-directed mutagenesis, also known as oligonucleotide-directed mutagenesis, is a technique that is used to intentionally modify a DNA sequence of a gene by introducing a site-specific mutation. The general method for producing the defined oligonucleotide mutation was developed from a combination of observations on nucleic acids, and it was in the 1978s where enzymatic extension was used as an approach to SDM, using oligonucleotides as primer (Hutchinson, et al., 1978). Since then, this technique has then been improved in efficiency in later studies which has stemmed from this initial mutagenesis experiment, and thus novel methods of inducing site-specific mutagenesis has been improved in later years.

Species with genomes which can be experimentally manipulated makes it possible for researchers to investigate the role played by genes in pathology. In organisms such as yeast and mice, the ability to add or delete specific genetic information leads to an unmatched level of precision in studies of gene function, and due to that more is known about their biological mechanisms than in any other within their respective taxonomic group (Urnov et al., 2010). A technique known as genome editing was originally used on Drosophila melanogaster promises to expand this capability to cells and entire organisms virtually any species. Genome editing enables well-organized and accurate genetic modification through the induction of double-strand breaks in a specific genomic sequence of choice; this is followed by the generation of desired changes during subsequent repair of the broken DNA. One way of doing genome editing is by use of Zinc finger nuclease (ZFN), which is a designed, sequences specific endonuclease that can be tailored to cut a user-chosen DNA target leading to deletion or addition of certain bases with the DNA strand (Urnov et al., 2010).

In 2012, it was discovered by Jinek et al [1], that a piece of RNA along with the Cas9 nuclease could be used to make cuts at any place in the DNA sequence. The greatest advantage of this method is that it is very easy to engineer pieces of RNA corresponding to the DNA sequence to be cut. Also, the Cas9 nuclease is easy to produce. This method provides a cheap, quick and easy way to make genetic changes in cells, and accelerate genetic research in the laboratory.

Gene editing is one of the most researched fields of molecular biology, as manipulated genes deem possible studying the specific genes and their effects. In the past, there were several attempts to manipulate gene function, including homologous recombination, RNA interference (RNAi), zinc-finger nucleases (ZFNs) and transcription-activator like effector nucleases (TALENs). These past approaches are expensive and time-consuming to engineer as opposed to Crispr Cas9, limiting their widespread use. What the Crispr Cas9 technique offers, and biologists desire, is specificity: the ability to target and study particular DNA sequences in the expanse of a genome with ease of preparation as used in Fu et al. (2014) and Korkmaz et al. (2016).

As much as life has evolved, the relationship between animals and humans has remained almost identical to each other. Dideoxynucleotide sequencing commonly called Sanger sequencing illustrates the way in which DNA sequencing has revolutionized molecular biology. In order to perform the sequencing, the first step it to convert double stranded DNA into single stranded DNA by applying a technique called annealing where the samples are heated to temperatures above 90°C, then the temperature is lowered, allowing the strands to separate and primers to join together. A Sanger reaction consists in a single stranded DNA, DNA polymerase, DNA primers and a mixture of ddNTP along with dCTP, dGTP, and dTTP.