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. 2022 Mar 28;28:353–362. doi: 10.1016/j.omtn.2022.03.021

Figure 5.

Figure 5

A model for enhancing target specificity and editing efficiency of en-Cas12a based on chimeric DNA-RNA-guided engineering

(Upper left) wt-crRNA guided wt-Cas12a system. In general, the wt-Cas12a effector can induce genetic mutations in target sequences but can also induce mutations in similar off-target sequences. (Bottom left) wt-crRNA-guided en-Cas12a system. Recognition of a target sequence is enhanced by an effector engineered by amino acid substitution, and thus the efficiency of inducing gene mutations is increased compared with that of the wt-Cas12a effector. However, due to the same principle as target sequence recognition, there is a problem in that the mutation induction efficiency of off-target sequences is also increased. (Upper right) Chimeric-crRNA-guided wt-Cas12a system. Effectively reduced off-target mutation induction efficiency when using a chimeric DNA-RNA guide with 8 nt DNA substituted at the 3′ end. However, the efficiency of mutation induction for the target nucleotide sequence in the genome is also reduced, and the efficiency is restored only when there is the action of nickase on the nucleotide sequence near the target. (Bottom right) Chimeric-crRNA-guided en-Cas12a system. This maximizes the target sequence indel ratio (%) and minimizes the off-target indel ratio (%) when using a chimeric DNA-RNA (8DNA)-guided en-Cas12a effector. It can induce more accurate and high-efficiency gene editing than wt-Cas12a on genomic DNA. PAM (TTTN) sequence for Cas12a effector is indicated by the yellow box. DNA cleavage points are indicated by red arrowheads, and the degree of cleavage is indicated by arrowhesd size according to the Cas12a activity. The wt-Cas12a and en-Cas12a effectors are shown in blue and brown, respectively. In the wt-crRNA and chimeric DNA-RNA guides, RNA is indicated in dark blue and nucleotides replaced with DNA in green.

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