During which will not turn brown (when

During the last five
years, enormous excitement has been witnessed with the discovery and utility of
the following three genome editing technologies: zinc finger nucleases
(ZFN), transcription activator-like
effector nucleases (TALEN)
and regularly interspaced
short palindromic repeats/CASPR associated protein 9 (CRISPR/Cas9). These genome editing
approaches make use of nucleases, which are used as scissors to create double
strand breaks (DSB) in DNA, and allow manipulation of the genome at the place
of DSB1 (for a Review, see Gaaj et al., 20131). However, of
the three nucleases, CRISPR/Cas9 attracted the maximum attention and has been
successfully used for gene editing in several plant and animal systems. An
improved version of CRISPR/Cas9 was also developed later in the form of CRISPR/Cpf1,
which had certain advantages over CRISPR/Cas92,3 (Zetche et al.,
2015; Zaidi et al., 2017).

     ZFN//TALEN/CRISPR-mediated genome editing has
been a preferred approach over transgenics, since no foreign gene is being
introduced, and only an existing gene is altered, using cells own machinery
involving homology-dependent repair (HDR) and non-homologous end joining
(NHEJ). The preferred HDR-dependent genome editing is,
however, limited by low efficiency arising from competition with NHEJ and from
the dependence of HDR on mitosis4 (Komor et al., 2016). Also, this technique
of genome editing does not allow an alteration of a specific existing base pair
in a DNA molecule in a predictable manner.

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     In
view of the fact that no foreign gene is inserted during genome editing, it has
been argued that products of genome editing involving nuclease-mediated
technologies like CRISPR/Cas9 should not be subjected to the regulatory system,
which is used in case of genetically modified organisms (GMOs) making
commercialization of the GMOs difficult. Consequently, a strain of ‘mushroom’ with
white buttons, which will not turn brown (when stored) was developed using
CRISPR and commercialized in USA without being subjected to the regulations
that are commonly applied to GMOs5,6 (Hall, 2016; Waltz, 2016); in
these edited mushrooms, one of the genes encoding PPO (polyphenol oxidase) that
is responsible for browning of mushrooms was altered, thus reducing the
quantity of PPO to 30%. A mutant corn that gave high yield under drought conditions
has also been developed through genome editing by DuPont and approved for
commercial cultivation, so that it may also be used for commercial cultivation
by the farmers and may hit the market soon7 (Shi et al. 2016).  

     Engineered CRISPR system contains the following two
components: (i) a guide RNA (gRNA
or sgRNA), and a (ii) a CRISPR-associated endonuclease (Cas protein).
The gRNA is a short
synthetic RNA composed of a scaffold sequence
necessary for Cas-binding and a ~20 nucleotide spacer (upstream of PAM sequence) that defines the genomic
target to be modified. In this manner, one can change the genomic target of the
Cas protein by simply changing the target sequence present in the gRNA.

 

     However, CRISPR/Cas9-mediated gene editing
can not be used for single specific base alteration in the genome and the
outcome of CASPR-mediated gene alteration can not be precisely predicted, since
a variety of products are obtained. Although Cas9 enzyme cuts DNA at the
desired location, the cells’ own DNA-repair systems fix the break. This can
create a variety of different edits in the genome, so that the desirable
alteration occurs at a frequency of not more than 5%. CRISPR-Cas9 also introduces
random insertions, deletions, translocations and other base-to-base conversions,
which is another limitation associated with CRISPR/Cas9 system.

     CRISPR/Cas is a prokaryotic adaptive immune
system that recognizes and degrades invasive foreign DNA/RNA (e.g., DNA/RNA
virus genome). A similar system called activation-induced cytidine deaminase
(AID) system also occurs in vertebrates, which is responsible not only for
protecting the vertebrate cells from invaders by causing alterations in the
genome of these invaders (like prokaryotic CRISPR/Cas), but also  generates antibody diversity through hypermutations
(C®U = C®T) in the variable
region of the immunoglobulin locus that produces antibodies. A hybrid system combining
these two systems a nuclease deficient prokaryotic CRISPR/Cas (rendering it
incapable of causing DSB) with vertebrate AID system was recently developed,
where CRISPR/Cas could produce s suitable target for cytidine deamination by
AID. This was described as Target-AID or CRISPR-AID (Fig. 1) and was used in
yeast, demonstrating that this can really be used for predictable gene editing
in eukaryotic systems8 (Nishida et al., 2016).