The (BE1) included the formation of undesired products due

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 cytosines (Cs) within the target window of 4-8 bases, which would allow base editing
of non-target cytosines also, in
addition to the target cytosine. The
enzyme UNG works during Base Excision Repair (BER) and therefore, will treat transitional edited base pair U:G (derived from C:G) as DNA damage, so that U of U:G base pair will be excised,
and U:G will not be able to produce T:A, leading to a failure of desired
conversion of C:G into T:A. 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
UNGwill be inhibited and will not be
able to excise U from
the U:G base pair, which
will be converted to T:A during DNA replication.
The editing efficiency of these 2nd generation base editors (BE2)
was improved (reaching a
maximum of ~20 and formation
of indels significantly reduced (<0.1%) over that obtained in 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 from 4-8 base pairs 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 nCas9), or replacement of amino acid histidine (H) by alanine at position 840 (H840A). nCas9 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 to a Streptococcus pyogenes nuclease deficient nickase Cas9n Cas9n(D10A), 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 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 (see later).           It was also shown that addition of another copy of UGI to BE3 further reduced the frequency of indels, so that BEs with more than one UGI were developed and were described as 4th 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).