For that were described as second generation, third generation

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 (