Another as 37-fold in case of HF-BE324

Another class of base
editors represented the so-called high-fidelity base editors (HF-BE), which
contained high fidelity Cas9 variants (HF-Cas9) including Cas9-HF1
with four mutations (N497A, R661A, Q695A and Q926A) and Cas9-HF2 with one
additional mutation (D1135A), thus conferring higher level of
specificity22,23. High fidelity HF-BE2 and
HF-BE3 were also developed and utilized for base-editing in
tyrosinase gene (tyr) in order to
examine the efficiency of these base editors. It was observed that the desired
base editing occurred and off-target activity was low, although proximal
off-target deamination was also observed, up to 30-40 bp away from the gRNA binding
site and from the PAM sequence. The base edited mice were also obtained through
transplantation of base edited embryos in surrogate mothers, suggesting no
embryonic toxicity during base editing. The mutant albino pups were obtained in
a frequency of 18.2% for gRNA1 and 63.6% for gRNA220. Often, with HF-BEs, C®T conversion in mouse embryos occurred with 100%
efficiency, associated
with reduction in off-target editing reaching as high as 37-fold in
case of HF-BE324 (Figures 8,9).