During yield under drought conditions has also been

During the last five years, enormous excitement has been witnessed with
the discovery of genome editing approaches involving sequence-specific designer
nucleases (ZFN, TALEN, CRISPR/Cas), which create double strand breaks in DNA (for a Review, see Gaaj et al., 20131). However, of the
three nucleases, CRISPR/Cas9
attracted the maximum attention for developing several plant and animal species
with desired genetic modifications through genome editing. An alternative for
Cas9 in the form of Cpf1 later became available giving birth to superior system
in the form of CRISPR/Cpf1, which has several advantages over CRSPR/Cas2,3
(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, since NHEJ competes
with it and creates high frequency of indels and off-site alterations during
genome editing. Also, 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 and only an
endogenous gene is altered, it has been argued that products of genome
editing technologies like
CRISPR/Cas9 should not be
subjected to the regulatory
system, which is used in
case of genetically
modified organisms (GMOs). This has made
commercialization of genome edited products easier at least in some countries. 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, a gene for 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).

     Sequence-specific designer CRISPR/Cas9 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. The gRNA iais designed
keeping in mind the sequence that needs to be altered, so that Cas9 binds the
the desired site and creates DSBs at specific desired site. In other words, the
genomic target for binding Cas protein and for creating alteration in a
specific region of the genome is decided by gRNA.

     However, outcome of CRISPR/Cas9-mediated
alteration in the genome is not precise at the individual nucleotide (base)
level, and therefore it can not be used for specific alterations at the level
of single specific base. In actual practice it has been noticed that a variety
of products are obtained and a selection needs to be exercised to obtain the
desired product, which is generally available 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.

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