Another as 37-fold in case of HF-BE324

Another class of baseeditors represented the so-called high-fidelity base editors (HF-BE), whichcontained high fidelity Cas9 variants (HF-Cas9) including Cas9-HF1with four mutations (N497A, R661A, Q695A and Q926A) and Cas9-HF2 with oneadditional mutation (D1135A), thus conferring higher level ofspecificity22,23.

High fidelity HF-BE2 andHF-BE3 were also developed and utilized for base-editing intyrosinase gene (tyr) in order toexamine the efficiency of these base editors. It was observed that the desiredbase editing occurred and off-target activity was low, although proximaloff-target deamination was also observed, up to 30-40 bp away from the gRNA bindingsite and from the PAM sequence. The base edited mice were also obtained throughtransplantation of base edited embryos in surrogate mothers, suggesting noembryonic toxicity during base editing. The mutant albino pups were obtained ina frequency of 18.2% for gRNA1 and 63.

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6% for gRNA220. Often, with HF-BEs, C®T conversion in mouse embryos occurred with 100%efficiency, associatedwith reduction in off-target editing reaching as high as 37-fold incase of HF-BE324 (Figures 8,9).


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