target gene, so in some cases this method can be less predictable and effective.
Zinc finger nucleases and TALENSs both induce double stranded breaks that
activate damage response pathways much similar to crispr but ZFN and TALEN
are composed of zinc finger protein or TALE respectively plus FokI fusion protein
while crispr consists of a guide RNA and cas9 protein and furthermore, crispr has
a lower cost, is more time efficient, utilises RNA instead of protein and can target
multiple sites(Carroll, 2014; Gaj et al., 2013; Wanyou et al., 2015). Cripsr has
already been proven to work in gene editing in a number of systems zebra fish,
mice, c. elegans, crops and etc.
Crispr cas has proven to work for targeted genome editing. Customised sgRNAs
were generated directing Cas9 alterations of endogenous genes in zebra fish
embryos and the frequency of altered alleles in single embryos was assessed
using a T7 endonuclease and furthermore to test the efficiency of the sgRNA:
Cas9 system in zebra fish, they constructed ten additional sgRNAs, each targeted
to another sequence in the fh gene and the remaining nine targeted to sites in
nine other endogenous genes. This showed that the sgRNA:Cas9 system
successfully targeted >80% of the sites tested in zebra fish (Hwang et al., 2013)
.Crispr has been successfully shown to work for multi-gene targeting in mice as
well. This may save time for generation of mice carrying mutations in multiple
genes because they are traditionally generated by sequential recombination in
embryonic stem cells and/or by time consuming inter-crossing of mice with single
mutations. CRISPR/Cas-mediated gene editing can permit the simultaneous
disruption of five genes (Tet1, 2, 3, Sry, Uty - 8 alleles) for mouse embryonic
stem cells with high efficiency. It was shown that coinjection of Cas9 mRNA and
single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice
with bialelic mutations in both genes worked with an efficiency of 80% (Wang et
al., 2013).Thus, the CRISPR/Cas system allows the one-step generation of
animals carrying mutations in multiple genes, an approach that will greatly
accelerate the study of functionally redundant genes and of epistatic gene
interactions.
Crispr cas can be used for designing experiments generating targeted loss of
function mutants. A generated vector expressing cas9 and sgRNA and an added
NLS to ensure the nuclear localisation, and a putative polymerase 3 promotor for
expression of the sgRNA was used as the basis for an experiment for C.Elegans
targeted loss of function (Friedland et al., 2013). It is known that crispr works
through double stranded breaks that can be repaired through the pathway of
nonhomologous end joining (NHEJ), which generates insertions and deletions
(indels) in the vicinity of the cleavage site(Cho, Kim, Kim, & Kim, 2013; Cong et
al., 2013).Therefore indels disrupting the coding sequences of genes would show
alleles that cause unique phenotypes. Crispr was shown to generate disruptions
in 53/66 (80.3%), 27/35 (77. 1%) and 13/72 (18.1%) of the F1 worms
screened(Friedland et al., 2013) indicating it works as a tool for generating loss
of function. Crispr has been shown to work to induce sequence-specific genome
editing in rice. This was facilitated by the design of two sgRNA, (SP1 and SP2),
which target different DNA strands of the rice OsPDS gene with a targeted
mutagenesis rate of 15%(Shan et al., 2013). Cloning and sequencing revealed
indels in the targeted OsPDS gene with the highest frequency of mutations were
obtained with a sgRNA with 20 nucleotides (nts) of sequence complementary to
the OsPDS-SP1 target site (P = 0.039)(Shan et al., 2013).
Crispr may help as a supplementary tool in studying human disease as it allows
for the more simplified production of genetically modified model species
monkeys for human research. Monkeys serve as an important model species for
studying human diseases but their research has been hindered by the difficulty
in producing monkeys with the desired mutations. Crispr cas has been shown to
achieve precise and simultaneous disruptions of two target genes (ppar-g and
Rag1) with no off target mutagenesis (Niu et al., 2014). This shows that the
simultaneous addition of cas9 mRNA and sgRNAs are a precise and simplified
way to target multiple genes. In addition, lentiviruses have already been used to
make successful transgenic monkeys(Niu et al., 2010) but they are not as
successful (Chan, 2013) as they have not been able to work successfully for
precise gene targeting as does crispr.
Cripsr is still in its infancy for human use and may require caution for its future.
At present, we dont know in scrupulous details the mechanism of crispr in
human zygotes because a serious knowledge gap remains in our understanding
of these mechanisms and in the efficiency and potential off-target effects of
using crispr which is evidenced by the attempt of Chinese researchers to use
crispr in tripronuclear (3PN) zygotes to cleave the endogenous -globin gene
(HBB) (Liang et al., 2015). The experiment demonstrated that the efficiency of
(HDR) for HBB was low and embryos would result in mosaics. Also, off-target
cleavages were produced which has already been seen (Fu et al., 2013) as well
as the endogenous delta-globin gene (HBD) interfering with exogenous donor
oligos leading to undesired mutations(Liang et al., 2015). This highlighted the
current knowledge gap and the need to be cautious and further improve the
fidelity and specificity of crispr before any clinical human trial can take place as it
is still in development and the edits will be permanent.
Furthermore, some have insisted on imposing a moratorium to discourage germ
line genome modifications for clinical applications in humans, in order to create
ethically responsible use for the technology (Cyranoski, 2015; Marshall, 2000;
Norman, 1983; Vogel, 2015). Others have suggested that this is pointless and
unenforceable as it will only drive this type of research underground (Hawkes,
2015) others see this type of research as a godsend and the future of modern
medicine as it allows the possible total eradication of genetic diseases that have
long existed in some families germ lines such as Huntingtons disease,duchenne
muscular dystrophy, fragile x syndrome, cystic fibrosis (Baltimore et al., 2015;
Doudna, 2015; Li et al., 2015; Pellagatti, Dolatshad, Valletta, & Boultwood, 2015;
Saayman, Ali, Morris, & Weinberg, 2015; Vasil'eva, Melino, & Barlev, 2015; Wu et
al., 2015) Others say we need to create forums where information can be spread
about the risks and rewards of using this technology and its applications and
implications so that practitioners can make informed decisions about how they
are using it and weigh up the risks and rewards and pros and cons. Encourage
the support of research to further improve the specific efficiency of crispr cas
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