Alongside CRISPR-Cas9 in site specific genome editing are ZFNs and TALENs. ZFNs are a DNA-binding motif assembled as ββα that utilizes a roughly thirty amino acid protein with DNA recognizing amino acids at the alpha helix (Gaj, Gersbach, & Barbas, 2013). These groups of amino acids usually recognize DNA in segments of three. This does not present much specificity in a genome. Therefore multiple DNA-binding motifs need to be combined to create specificity and result in highly specific polypeptides of repeating zinc finger complexes that recognize nearly eighteen base pair lengths. This structure results in relatively large molecules and the size increases due to ZFNs main nucleases only functioning as dimers. A dsDNA break requires two
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Injecting one-cell-stage embryos with Cas9 mRNA and sgRNAs immediately after manual fertilization resulted in 10 of 29 pregnancies in surrogate mothers. Further, there were no off-target mutations, which is the biggest concern with CRISPR-Cas9 in these kinds of experiments. Off-target mutations are mutations caused by CRISPR-Cas9 being tolerable of mismatches in the crRNA near the PAM sequence, resulting in Cas9 activity in unwanted areas of DNA. ZFN and TALEN methods are not feasible in monkeys so far; however, they are amenable to some target animals (Niu et al., 2014). A similar experiment consisting of Cynomolgus monkeys was attempted with TALENS. After injecting TALENS at the single cell embryo stage of fifty-four samples only six host females became pregnant. Moreover to reach 50% efficiency multiple variants were needed in tandem, which produced significant mosaicism, an individual with varying genetic data, at the target locus and off-target effects in the form of point mutations, many of which were within the targeted exon (Liu et al., 2014). While the CRISPR system also produced mosaicism …show more content…
In turn, the risk of potentially disrupting these species outside the laboratory setting has been increased. For example, many labs have developed gene drive systems, which install a Cas9 system into the genetic code of an organism that is able to knock out the undesired gene upon inheritance and use the gene drive as a template of repair to replace the cut strand. This application has the possibility of removing a gene from a population almost entirely or inserting a desired gene, which creates a critical issue containing these organisms in the laboratory setting and minimizing unintended effects (DiCarlo, Chavez, Dietz, Esvelt, & Church, 2015). One such drive being researched in Florida seeks to eliminate dengue fever in a population of mosquitoes. A leak of these organisms such as these could have dramatic effects on the local ecosystem, but it also brings the question of use up to people and governments in the region. The advances in CRISPR technology and the rate at which they are occurring has caused a surge of interest from the public and scientific community regarding genetically modifying organisms on Earth to better suit the needs of people and on potential human therapeutics.
Injecting one-cell-stage embryos with Cas9 mRNA and sgRNAs immediately after manual fertilization resulted in 10 of 29 pregnancies in surrogate mothers. Further, there were no off-target mutations, which is the biggest concern with CRISPR-Cas9 in these kinds of experiments. Off-target mutations are mutations caused by CRISPR-Cas9 being tolerable of mismatches in the crRNA near the PAM sequence, resulting in Cas9 activity in unwanted areas of DNA. ZFN and TALEN methods are not feasible in monkeys so far; however, they are amenable to some target animals (Niu et al., 2014). A similar experiment consisting of Cynomolgus monkeys was attempted with TALENS. After injecting TALENS at the single cell embryo stage of fifty-four samples only six host females became pregnant. Moreover to reach 50% efficiency multiple variants were needed in tandem, which produced significant mosaicism, an individual with varying genetic data, at the target locus and off-target effects in the form of point mutations, many of which were within the targeted exon (Liu et al., 2014). While the CRISPR system also produced mosaicism …show more content…
In turn, the risk of potentially disrupting these species outside the laboratory setting has been increased. For example, many labs have developed gene drive systems, which install a Cas9 system into the genetic code of an organism that is able to knock out the undesired gene upon inheritance and use the gene drive as a template of repair to replace the cut strand. This application has the possibility of removing a gene from a population almost entirely or inserting a desired gene, which creates a critical issue containing these organisms in the laboratory setting and minimizing unintended effects (DiCarlo, Chavez, Dietz, Esvelt, & Church, 2015). One such drive being researched in Florida seeks to eliminate dengue fever in a population of mosquitoes. A leak of these organisms such as these could have dramatic effects on the local ecosystem, but it also brings the question of use up to people and governments in the region. The advances in CRISPR technology and the rate at which they are occurring has caused a surge of interest from the public and scientific community regarding genetically modifying organisms on Earth to better suit the needs of people and on potential human therapeutics.