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 (<0.1%) both in BE1 and  BE2, since the DNA was not directly cleaved as in case of CRTISPR-mediated genome editing. The second problem of the occurrence of more than one Cs in the editing window was partly resolved by reducing the size of editing window to 1 or 2 base pairs (see later).      The next stage of improvement of base editors was achieved by converting dCas9 to a nickase through replacement of either amino acid aspartate (D) by alanine (A) at position 10 (D10A; also described as Cas9n), or replacement of amino acid histidine (H) by alanine at position 840 (H840A). Cas9n and H840A both produce nicks in opposite strands, and have been suitably utilized in single base gene editing14 (Ran et al., 2013). For instance D10A mutant of Cas9 retains a domain that generates a single strand DNA nick in the non-target strand instead of creating double strand breaks at the desired site; this would simulate mismatch repair, so that a unmodified opposite DNA strand would mimic a DNA strand undergoing synthesis, where the strand containing the edited base is used as a template (C ®U; Fig. 4), taking U as T. Therefore, BE3 had the following three components: (i) an AID/APOBEC1 deaminase, that was fused through a 16–amino acid linker to (ii) a Streptococcus pyogenes nickase Cas9n Cas9n(D10A), which was first disabled for its nuclease function and was later converted into a nickase (Cas9n) and (iii) a UGI that was linked to Cas9n through a 4 amino acids linker. The importance of UGI in base editing was demonstrated by showing that the UGI-deleted BE3 (BE3-?UGI ) was less competent in base editing compared to original BE3, and produced not only lower frequency of desired C®T editing, but also produced a higher frequency of unwanted indels. A number of improved BE3 variants were also developed (Table 2), which resulted in much more efficient conversion of the G:U intermediate to desired A:U and A:T products4,11 (Komor et al., 2016, 2017).      Another problem associated with BE1 and BE2 was the occurrence of more than one Cs within the base editing activity window, so that the cytosine deaminase will convert even a non-targeted C into U. This problem was overcome by the development of BE3 with SpCas9 (NGG), where even the non-NGG PAM sequence could be used for base editing.      It was also shown that addition of another copy of UGI to BE3 further reduced the frequency of indels, so that BEs were later improved by having more than one copy of UGI associated with Cas9n and cytosine deaminase. These were described as fourth generation base editors, the BE4, which were found to be more efficient (Wang et al., 2017).  BE4 or SaBE4 were further improved by adding Gam to the cassette, so that the use of BE4-Gam resulted in a further 1.5 to 2.0 fold decrease in the indel frequency (Table 1).