Applications have been described as molecular machines, which

Applications of Base Editing

 

A number
of BEs and ABEs have been used for a wide variety of applications, including
plant genome editing, in vivo
mammalian genome editing, targeted mutagenesis, and knockout studies (1, 7–9, 12–19).
Some of these applications including future possibilities will be discussed.

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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
disease-causing point mutations. Many of these mutations, each involving single
base alteration cause serious diseases, ranging from genetic blindness to
sickle-cell anemia to metabolic disorders to cystic fibrosis, for which no treatments
are available at present. It has been estimated that approximately half of the
32,000 disease-associated point mutations already identified by researchers are
a change from G:C to A:T, which can be corrected by BE3, BE4 and their
different variants. 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.

     The BEs and ABEs developed by David Liu and
his team have been described as molecular machines, which make the desired and predictable
genetic change for treatment of a diseases. 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%. Similarly, using human
cells, mutation in a gene associated with a cancer could be corrected with up
to 7.6% efficiency. These corrections could not be possible using standard
CRISPR­Cas9 method. Therefore,
“base editors” and not CRISPR/Cas9, can correct many harmful point mutations
that are associated with a number of diseases, for which no treatments are
available at present. In order to make it a reality
for human health care, delivery of this machine and its safety and side effects
are the questions, which are being addressed.

   Researchers in China also reported
that they had used Liu’s base editor to correct a single base mutation or point
mutation, for a blood disorder in human embryos (The embryos were not allowed
to develop further).

     A team in Korea used
mouse zygotes as a model system and  targeted the Dmd or Tyr gene.
F0 mice showed nonsense
mutations with an efficiency of 44–57% and allelic frequencies of up to 100%,
demonstrating an efficient method to generate mice with targeted point
mutations (Kim et al., 2017).

(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 (target-activation-induced
cytidine deaminase) 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,…..

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