PAM were used, one carrying sgRNA, second carrying BE3

PAM site and size of targeting window as two limitations

 

As mentioned earlier, the occurrence of a downstream
PAM sequence in association with target protospacer sequence is necessary for
dCas9 or nCas9 to bind (with the help of gRNA) and cleave the target DNA
sequence for base editing. In the absence of PAM, targeted base editing will
not be possible. However, the PAM sequence associated with Cas in different
bacteria differ, although the canonical PAM sequence is 5′-NGG-3′ (Figure 1),
which is associated with SpCas9 nuclease from Steptococcus pyogenes. This limits the number of targeted genome
sites, which can be efficiently targeted by BE3 and its variants, because many
sites would lack NGG PAM at the desired location. Attempts, therefore, have
been made to improve BE3 variants through engineering Cas9, so that CRISPR/Cas9
may be able to conduct gene editing at any desired genome location (using
non-canonical-PAM) and to reduce the editing window to 1-2 nucleotides through
introducing appropriate mutations in Cas9, to avoid off-site editing (Table 2).

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For instance, SpCas9 was replaced by SaCas9 (from Staphylococcus aureus), which is considerably smaller and is
associated with NNGRRT PAM, and has been successfully used in several studies.

The editing efficiency in several of these BE3 and BE4 editors was as high as
~50%.

 

Enhanced
BEs (eBE-S1, eBE-S3) and high fidelity BEs (HF-BE)

 

Two
other classes of improved BEs included enhanced base editors (eBEs) and high
fidelity BEs (HF-BEs). An enhanced BE (eBE) involved designing of the
following two systems, and their expression was tested in FT292 and HeLa cells:
(i) In one system, three independent vectors were used, one carrying sgRNA,
second carrying BE3 and the third carrying UGI gene (Figure 7a); this would
allow co-expression of BE3 and sgRNA with free UGI in trans; expression level of UGI was also manipulated to study the
relationship between the level of UGI and precision in base editing; it was
observed that the ratio of C®T conversion
to indel frequency was positively correlated and that at high level of UGI, there
was six fold increase in the proportion of C®T conversion relative to indels; unwanted C®A and C®G substitutions relative to C®T conversion also declined. (ii) In the other system, sgRNA was
allowed to coexpress with BE3 carrying one  (eBE-S1) or three (eBE-S3) additional copies
of 2A-UGI sequence (Figure 7b). In one study, five different sgRNAs were independently
used for targeting five different loci in FT293 cells; both enhanced BE3s (eBE3)
exhibited a decline in indel frequency and an improvement in the frequency of targeted
C®T, when compared with original BE3
carrying only one copy of UGI19
(Figure 5). It has been recommended
that the approach should be combined with altered PAM and nCas to get a very high
level of precision19. The
approach has since been
deployed in a range of organisms,
from wheat to zebrafish and mice. 

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