Multiplexed inactivation of endogenous genes by base-editor-mediated multi-stop system

One of the major hurdles in CRISPR application is the present of unwanted off-target mutations. To achieve multiple gene targeting with minimal off-target side-effects, we evaluated the fidelity of hA3A-eBE-Y130F system by targeting two genes (Tyr and Crygc) using GOIT method. The results indicated that higher amount of SNVs present in hA3A-eBE-Y130F injected cells than that of Cre or eY injected only, but the indel ratio remains the same. The increases in SNVs resembles previously report that base editing system could lead to unwanted mutations in genomic scale due to the basal activity of coupled enzyme, and the de-activated Cas protein will not induce double strand breaks which will give the rise of indels. Among all the SNVs detected, cells injected with eY is most likely to induce C->T/G->A base conversion comparing with Cre injected only.