Applications including BEs and ABEs can correct each

Applications
of Base Editing

 

A variety of BEs and ABEs have the potential of being
used for a wide variety of applications including human healthcare and crop
improvement. Some of the initial studies conducted during 2016-17 demonstrated
this potential. Studies have been conducted using mouse, Xenopus and zebrafish as human models for the study of successful use
of BEs and ABEs. Similarly, there are several reports of crops like rice,
wheat, maize and timato, where specific genes have been edited using BEs,
leading to the development of mutant plants with desirable traits. In some
cases, these mutant plants have been described as transgenic plants, but we
will avoid calling them transgenic, since no transgenes have been inserted in
the genome, and only genes have been altered, making them equivalent to mutant
plants.

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(a) Application
of Base Editing in Human Health

 

As shown in this article, the base
editors including BEs and ABEs can
correct each of the the
following four “transition” mutations; C®T, T®C, A® G, or G® A, which together account for almost two-thirds of all point mutations that cause diseases. A number of
diseases including sickle cell anemia, genetic blindness, cystic fibrosis and
several metabolic disorders, for which no treatment is currently available, are
associated with single nucleotide polymorphisms (SNPs) suggesting that editing
at single base level may ameliorate patients from the disease. A large number
of diseases are caused by one of the above four transitional changes, although
disease may also be caused due to transversions (a purine replaced by a
pyrimidine or vice versa). About 50% of 32,000 disease causing point mutations
are known to be due to a change
from G:C to A:T which can be corrected by different variants of BE3/BE4. These
are also diseases, which involve changes from A:T to
G:C, which can be
corrected using ABEs. These base editors could help in the future development of
gene-therapy approaches (Gaudelli et al., 2017)9. Additional
research is, however, needed to enable
BEs and ABEs to target as much of the genome as possible.  In view of this, these BEs and ABEs have been
described as molecular machines by David Lui and his team

      Theat
base editors can be used for correction of single base genetic defects has been
demonstrated by using several models including mouse, zebrafish and Xenomus. For instance, using mouse cells grown in culture, it
has been shown that the mutations associated with Alzheimer’s disease can be
corrected using BEs with an efficiency of up to 75%. Human cells have also been used to provide
proof of the concept that some of the cancers caused by single base alterations
can be treated through base editing.  Standard CRISPR­Cas9 method of genoime editing may not help in many of
these cases. However, it is recognized that a safe and reliable delivery
of a molecular machine with no side
effects. These concerns are already being addressed, so that in the near future
gen thery based on base editors will become available, although it may take
time for them to reach the doctors clinics..

      Several reports are available, where
corrections of individuasl genes has been demonstrated even at the organismal
levels in model systems like mouse, zebrafish and Xenopus, where altered
emryoes were transplanted in pesudopegnant surrogate mothers and mutant
offspring obtained. The gene tyr
encoding tyrosine pigment causing albinism has been successfully used in
several studies (Kim et al., 2017). In China, a single base mutation for a blood disorder was
corrected in human embryos using a molecular machine in the form of base
editor, although the  embryos were not
allowed to develop further.

 

(2) Application of
Base Editing in Crop Improvement

 

Examples
of successful base editing are also available
in plants. In most cases, a BE3 variant with nCas9 nickase fused with a cytidine deaminase and
a UGI was used for base editing. Since delivery of template DNA can
sometimes be a problem in plants, a Target-AID
was used as cytidine deaminase
(Shimatani et al., 2016),
and the fusion product was codon optimized for plants (cereals); these base
editors were, therefore, described as plant
base editor =
PBE (Zong et al. (2017).

 

The crops, which were used for base editing
included cereals (rice, wheat ad maize (Zong et al., 2017), rice and tomato (Shimatani et al., 2017)

 

     These examples include the following: (i) In a study in rice, nCas9 nickase was
fused with a cytidine
deaminase enzyme and a UGI
to generate targeted mutations. The
BE3 cassette was inserted in pCXUN vector to generate
pCXUN-BE3, which had the ability to target a specified locus, when a gRNA
molecule is simultaneously expressed (….). Expression cassette of a gRNA under
the control of the rice U3 promoter was inserted into the PmeI site of pCXUN-BE3.

  

Three targets were chosen: one target (P2) in the OsSBEIIb gene, which
encodes a phytoene desaturase, and two targets (S3 and S5) in the gene OsSB,
which encodes a starch branching enzyme IIb.
The vectors were delivered into rice calli through Agrobacterium- mediated
transformation. The base-editing vectors
demonstrated their feasibility and efficacy (Li et al., 2017). Base editing was
successful at all the three loci with efficiency much higher than obtained
using CRISP/Cas9 system.   Zong et al. (2017)
 

      (ii) In another study, Zong et al. (2017) used two
plant base editors (PBE) carrying CASPR-nCas9 -cytidine deaminase (APOBEC1)
fusion proteins, namely nCas9-PBE and dCas9-PBE (Fig…). These were successfully
used for base editing in rice, wheat and maize with frequencies of individual
cytosine editing ranging from 5% to 32.5%,
with no  associated indels.  (iii)
In maize….(iv) In tomato,