Sio1 Case Study

From the science community perspective, the CRISPR-Cas system could reduce or even eliminate many of the difficulties researchers face when gene editing such as cost, duration and accuracy. Prior to CRISPR-Cas, gene editing was performed in “big labs” with experts

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.

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 first paper, “Cis Regulatory Effects on A-to-I RNA Editing in Related Drosophila Species,” compares Adenosine-to-inosine (A-to-I) RNA Editing between two Drosophila species: D. melanogaster and D. sechellia. The primary interest of this study is the regulation of RNA editing and whether this regulation is determined predominantly by

The editing site “destabilized the open state” by making the open duration become much steeper and quicker compared to the genomic version of the clone. This incurs a decrease in the probability of the open channels staying in their open state, and prevents action potentials from

This mutagenesis kit can make point mutations, replace certain amino acids, and delete or insert adjacent amino acids, making it an incredibly powerful tool. First, the mutant strand is synthesized by denaturing the DNA template and anneal the mutagenic primer(s) and then incorporated using DNA polymerase. A mutated plasmid is created and then digested by Dpn I endonuclease and transformed into competent cells for cell nick repair (Agilent, n.d.).

These small dsRNAs are the molecules loaded into the RISC to guide the complex to the target genes. The tiny dsRNAs act as escorts by using Watson-Crick base-pairing. Ultimately, the dicer enzyme facilitates the activation of the RNA-induced silencing complex. It belongs to the RNase III endo-ribonuclease enzyme family because it is a helicase with an RNase motif. In this experiment we tested to see if making a mutation in the dicer enzyme would inhibit its ability to cleave large dsRNAs into smaller ones. If the mutation does inhibit, it will cause the RNA interference pathway to come to a halt. Without RNA interference, the human body could stop producing oncogenes or tumor repressors. This could eventually result in

(…) Researchers can edit genes by ridding the faulty genes and replacing the lettering(coding) with the CRISPR-Cas9.

There have been many studies conducted using RNAi but very few have been conducted with using the Danio rerio, the zebrafish. However, the zebrafish is ideal for this study due to the fact that 70% of human genes share at least one obvious zebrafish orthologue. (Kerstin Howe, et. al, 2014) Furthermore, the zebrafish is one of the few species that posses the GABRB3 gene, which is linked to Aspergers Syndrome. The GABRB3 gene regulates the functioning of a neurotransmitter called gamma-aminobutyric acid. It has been said that the RNAi can be used to knock down the function of the gene, therefore “silencing” it. (Gopalakrishna; Slack; 2002)

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

Since shRNA does not interact with the Drosha/DGCR8 microprocessor complex, this RNA must be designed with care in order to achieve correct downstream processing and gene suppression.1 Proper dosage of shRNAs is another factor to consider, as it has been found that they may saturate the endogenous pathway and disrupt natural miRNAs.4

1998). Interestingly, the phenomenon that the introduction of double-stranded RNAs “quelled” the expression of endogenous genes contain homologous sequences had been observed and even utilized as a tool to knock down gene expression by researchers long before the underlying RNAi mechanism was identified and separated from the antisense silencing by Fire and Mello in 1998 (Fire et al. 1998). They consistently found that the introduction of double-stranded RNAs (dsRNAs) was 100 or more folds effective than the single-stranded “sense” or “antisense” RNAs (ssRNA) to silence the complement gene expression (Fire et al. 1998; Kennerdell and Carthew 1998). Moreover, the dsRNA mediated gene silencing can be transferred between adjacent cells, and even inherited through multiple rounds of cell division (Fire et al. 1998). Therefore, it is proposed that this so called “RNA interference” is an inheritable and transferrable process that involves a catalytic process, which differs from the previously known antisense silencing that ssRNAs directly bind to the complement mRNA targets to repress translation by blocking ribosome access. Subsequently, Tuschl and co-workers demonstrated that RNAi mechanism exists in mammalian cell lines for the introduction of synthetic

In 2011 Li et all published a journal article in Science describing a process that could have big consequences in the field of cellular biology and genetics. Although there are two known mechanism of post transcriptional RNA editing: adenosine turning into inosine (which during translation is interpreted as guanine) (A > I) and cytosine to uracil deamination (C > U), there were very few instances known where this was happening in a coding region that could affect translation and until now it was considered that DNA is faithfully translated into protein through RNA intermediates according to the central dogma of biology. In the paper published in Science the group claimed that they had identified upward of 10000 RNA/DNA mismatches many of which can produce changes in the protein sequence. The methodology used in the original paper was to sequence the mRNA expressed by an individual and compare it to the sequenced DNA. They called the differences between the mRNA sequence and DNA sequence RNA-DNA differences (RDD).

There was no significant difference between the single treatment with 10 µg/ml or 50 µg/ml of each dsRNA and the mixture formed of 10 µg/ml gyrase A and 10 µg/ml gyrase B dsRNAs and the mixture formed of 10 µg/ml rpo B1 and 10 µg/ml rpo B2 dsRNAs

The utilization of effector combinations can significantly extend the collection of genome designing modalities achievable utilizing CRISPR-Cas9. For instance, an assortment of proteins or RNAs can be fastened to CRISPR-Cas9 to adjust translation conditions of particular genomic loci, screen chromatin states, or even rework the three-dimensional association of the genome.