During consists of the nuclease Cas9 for creating a

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 (DSB) in
DNA (for a
Review, see Ref # 1). 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 a superior
system in the form of CRISPR/Cpf1, which has several advantages over CRSPR/Cas2,3.


genome editing has been a preferred approach over transgenics, since
no foreign
gene is being introduced, and only an existing gene is altered,
using cell’s own machinery. Therefore, 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 countries4. As an example, 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.

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In these edited mushrooms, a gene for PPO (polyphenol oxidase) that causes
of mushrooms
was altered, thus reducing the quantity of PPO to 30%. A
mutant waxy corn that gave high yield under drought conditions has also been
developed through genome editing by DuPont; this genome edited waxy corn was also approved
in USA for commercial cultivation, and may become available to
the farmers for commercial cultivation within the next few years7.

      The popular CRISPR/Cas9 system for genome
editing consists of the nuclease Cas9 for creating a double strand break (DSB)
at the target site, which is recognized with the help of an associated
synthetic guide RNA (gRNA), which is programmable and is designed on the basis
of target sequence that is intended to be edited. The sgRNA consists of a scaffold sequence,
which facilitates DNA binding of Cas9, and a ~20 nucleotide protospacer that is
complimentary to the sequence to be edited. This 20-nucleotide protospacer
needs to be so designed that it should lie upstream of a characteristic PAM (protospacer adjacent motif),
which differs in different microbes).  The canonical PAM is 5′-NGG-3′ (Figure 1), which is associated with the endogenous
Cas9 nuclease of Streptococcus pyroenes, so
that the corresponding Cas is designated as SpCas9. In the absence of PAM, genome editing may
not take place.

      The genome editing due to CRISPR/Cas9 is known
to have low efficiency, since NHEJ competes with the preferred HDR-dependent
genome editing. It also 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 or RNA molecule in a predictable manner. 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.