For series of base editors that were described

For the
purpose of precise single base editing, a number of plasmids called base
editors (BE) were developed during 2016-17. These plasmids facilitate base
editing (a transition) involving conversion of cytosine into uracil (Fig. 3),
leading to replacement of cytosine/guanine (C:G) base pair by thymine/adenine
(T:A) base pair. Since these base editors were meant for alteration of cytosine
only, these could be better named as cytosine base editors (CBE) as against
adenine base editors (ABE) that were developed for A®I(G) conversion later in 2017 (I = inosine).

     The first-generation C®U base editors (BE1) were developed using the rat cytidine
deaminase AID/APOBEC1 connected to a disabled Cas9 (dCas9) via a 16 base XTEN
linker4 (Komor et al. 2016).
AID/APOBECs (activation
induced deaminase/ apolipoprotein B mRNA editing enzyme, catalytic
polypeptide-like) used in this study represent a family of naturally occurring
cytidine deaminases, which use single-stranded DNA/RNA as a substrate11
(Knisbacher et al. 2016). The
members of AID/APOBEC family were combined with the CRISPR/dCas9 system to
perform targeted base editing. This combination improved CRISPR/Cas9-mediated
gene editing at single base precision, thus greatly enhancing its utility. The
original requirements for single base editing included the following
components: (i) a disabled Cas9 (dCas9) fused to a cytidine deaminase; (ii) a gRNA that helps dCas9 to target a specific locus associated with
a protospacer adjacent motif (PAM) sequence available ~18-20 base pairs downstream, and (iii) a target cytosine within a window of positions 4-8. These first
generation base editors (BE1) were further improved leading to the development
of a series of base editors that were described as second generation, third
generation and fourth generation base editors12 (BE2, BE3, BE4)
(Table 1). In each case, high-throughput DNA sequencing (HTS) was used to quantify base
editing efficiency. Digenome seq (sequencing of digested DNA) was also used for
assessment of off-target effects in human cells13 (Kim, D et al.

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Improvement of BEs
using Uracil N-Glycolase Inhibitors (UGI)


The major problem with
the first generation base editors (BE1) included the formation of undesired products
due to the following two reasons: (i) frequent removal of uracil by cellular N-glycosylase
(UNG) and (ii) possible occurrence of more than one Cs within the base editing
activity window of 4-8 bases, permitting base editing of non-target cytosines possible.
The enzyme UNG works
during Base Excision Repair (BER) and therefore, will identify transitional edited
base pair G:U as DNA damage and will excise U in G:U base pair, which is used
for the conversion of G:C into T:A base pair. Keeping this in view and in order
to increase in vivo editing
efficiency, second generation base editors (BE2) were developed, which carried
a uracil glycosylase inhibitor (UGI) fused with dCas9, so that the enzyme UNG
will not be able to excise U from the G:U base pair. The editing efficiency of these
second-generation base editors (BE2) was three-fold that of BE1 reaching a
maximum of ~20%; indel formation was very low (

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