The First Genome Editing Methods Essay

When scientists know what gene they want to manipulate they 'introduce double stranded DNA, or dsRNA to the cell.' The dsRNA produce small double-stranded interfering RNAs, or siRNA into the cytoplasm of the cell.5 from this the expression of the gene decreases drastically but does not get entirely eliminated, therefore showing the role of the targeted gene.

Long dsRNA s bind to Dicer to cut into smaller nucleotide fragments/segments. siRNA unwinds when it is catalysed by Argonaut. One strand remained with the Argonaut is the guide strand. The RNA guide strand with Argonaut and other proteins are combined into RNA induced silencing complex (RISC) and the other passenger RNA strand is released into nucleotides.

When the guide RNA finds its target within the viral genome, Cas9 cuts the DNA, incapacitating the

It may be noted that Sir2 functions as a silencer of large chromosomal regions, while Hst1 serves to repress specific genes. In addition, the regions that Sir2 silences rely on it interacting with different sets of cofactors; functional Sir2 cannot silence at the HM locus without Sir3 or Sir4, and it cannot silence at rDNA without Net1 or Cdc14.

The reduction can occur through genetic modification or treatment with a reagent such as short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript. Gene knockdown or RNA interference (RNAi) is a natural process which cells use to turn down, or silence the activity of specific genes. It is a biological process where RNA molecules inhibit gene expression, which is caused by the destruction of specific mRNA molecules. RNAi destroys the molecular messengers that carry information, which is coded in genes to the cell’s protein factories. The molecular messengers that are being destroyed are called messenger RNAs (mRNAs) which are destroyed by the RNAi molecules as they bind to the mRNA and deactivate the mRNA, so stop it from producing polypeptide, these mRNAs are used to carry out a critical function, without this a gene is essentially inactive. The double-stranded RNA molecules that trigger RNAi get cut into small fragments by the enzyme called ‘Dicer’. These small fragments serve as guides which lead the cells RNAi machinery to mRNAs that match the genetic sequence of the fragments. After this, the machinery then slices these cellular mRNAs, which effectively destroy their messages and shut off the corresponding gene. RNAi exists in many living things, including single-celled organisms, plants and

This article discusses scientists from Oregon Health and Science University who successfully edited the DNA of human embryos to eliminate a mutant gene of a heritable heart condition, using gene editing technology known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). They focused on an inherited heart disease called hypertrophic cardiomyopathy, which is the potential thickening of the heart muscle. The procedure consisted of using molecular scissors to cut out the mutation in the sperm right before it fertilized the egg. The embryo then repaired the cut in the sperm’s genetic material by copying the egg’s healthy gene, leaving the embryo free of a genetic disease that would otherwise be passed down by the father. It

The siRNA molecule is the product of the RNA interference (RNAi) pathway. The starting molecule in the pathway is a long “triggering” double stranded RNA (dsRNA). The dsRNA is formed from either an RNAi molecule inserted into the cell or a siRNA which complexes with a specific strand of messenger RNA

In Archetype II lncRNA works to titrate proteins and protein factors away from chromatins, yet titrating them into nuclear subdomains while serving as decoys. This is common in miRNA sites where lncRNA appears as decoys. Acting as guides in Archetype III, lncRNA functions in either cis or trans regions to assist in the recruitment of chromatin enzymes for target genes. In cis, lncRNA occurs in co-transcriptionally. This suggests that lncRNAs acts as tethers. However, lncRNA are known to also act in trans when searching for target sites. This determines the importance chromosomal looping plays in cis effects (Rinn and Chang, 2012). During Archetype IV lncRNA forms ribonucleoproteins by acting as scaffolds (Kung, Colognori and Lee, 2013). Additionally, lncRNA in the regulation of gene expression was recent discovered in Xist, a regulatory transcript found in the X chromosome that mediates the silencing of gene expression (Malecová and Morris, 2010). According to Nagano and Fraser (2011), lncRNA is critical in genomic reprogramming. For instance, somatic cells that are reconditioned to induced pluripotent stem cells. LncRNA can also act as a post-transcriptional regulation in Mrna. A lncRNA known as Gomafu/MIAT hinders the formation of spliceosome and inhibits the splicing of one of the subsets involved in mRNA (Kung, Colognori and Lee, 2013).

Once dsRNA is injected it binds to Dicer, an endonuclease protein that cuts RNA into short segments of 21 nucleotides long that are siRNA or micro RNAs. The short dsRNA binds to an argonaute protein and a guide strand is selected. The RNA, argonaute protein, and other proteins are called the RNA Induced Silencing Complex (RISC). Micro RNA’s help guide RISC to messenger RNA’s. The seed, part of the micro RNA, pairs with a target messenger RNA. Targeting by a micro RNA can lead to messenger RNA to be degraded or translation from being inhibited. Antisense RNA alone is not sufficient to evoke an RNAi response because the RNA needs to go through many steps with the help of other proteins to be able to have the ability to achieve RNAi.

c. The results were analyzed by 3 steps. First of all, specific regions of the genes were PCR amplified and visualized on the gel. Secondy, T7EN1 cleavage assay were used to detect sgRNA:Cas9 mediated on target cleavage of specific genes. Finally, PCR amplified regions were cloned and sequenced to determine the deletions or insertions.

Recent studies suggest that genome editing of germ line cells is an effective strategy for mutated gene correction in sperms and oocytes for the inhibition of onset of inherited disorder. The first time, germ line genome editing in human by CRISPR/Cas9 system , was carried out to correct mutation of HBB (Beta globin) gene in zygote of β-thalassemia patients by homologous DNA recombination (HDR) [80, 81]. Embryonic genome editing utilized in the treatment of such monogenic diseases prior to birth is a rational use of embryonic genome editing in germ line cells. Homologous DNA recombination by traditional gene editing techniques in mammalian eggs has a low rate (<10%) in term of

Since the discovery of the small RNA lin-4 in 1993 (Lee et al., 1993; Wightman et al., 1993), it has become evident that the miRNA machinery regulates many proteins and almost all cellular pathways (Pasquinelli et al., 2005; Stark et al., 2007; Filipowicz et al., 2008; Friedman et al., 2009). miRNAs are 20-23 nucleotide (nt) long RNA sequences encoded by genomic DNA (Bartel, 2004). After transcription, the RNA sequence destined to become a miRNA folds into a hairpin structure called the pri-miRNA. Pri-miRNAs are cleaved by the Drosha-Pasha complex in the nucleus and become ~70-nucleotide pre-miRNAs (He & Hannon, 2004), which are transported to the cytoplasm by Exportin 5. There, it is cleaved a second time by Dicer-TRBP complex to yield a 20 base pair (bp) double stranded sequence. The two strands are then separated, and one is degraded while the other is assembled into the miRNA-induced silencing complex (miRISC) (Filipowicz et al., 2008). Other components of miRISC are Argonaute proteins and other regulatory proteins such as fragile X-mental retardation protein (FMRP) (Jin et al., 2004a; Filipowicz et al., 2008). Mature miRNAs bind to seed regions in the 3 ' UTR of target mRNAs to either suppress their translation or mark them for degradation (Pasquinelli et al., 2005; Behm-Ansmant et al., 2006; Filipowicz et al., 2008).

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).

Next, crRNA biogenesis takes place in two stages. First, the CRISPR array and the Cas gene are transcribed respectively into a single pre-crRNA and Cas proteins. In this process, different types of CRISPR systems are unique in their Cas proteins they encode. Specifically, type II

In Elbashir, Lendeckel, and Tuschl’s article, they explore how 21- and 22-nucleotides RNAs mediate RNA interference by using an in vitro Drosophila system; this article is still referenced today as an authoritative source for RNAi research They show that the