Fig. 2: p53DD increased PE efficiency for precise insertion and deletion at endogenous loci. | Nature Communications

Fig. 2: p53DD increased PE efficiency for precise insertion and deletion at endogenous loci.

From: Transient inhibition of p53 enhances prime editing and cytosine base-editing efficiencies in human pluripotent stem cells

Fig. 2

a Schematics of pegRNA design and PE tools for precise “CTT” or “Loxp” insertion or “GT” deletion at HEK3 locus in H1 hESCs. The PegRNA was designed to include a protospacer sequence that targets the HEK3 locus, a RT template of the 3′ extension was designed to include the edit: CTT (3 bp) or LoxP (34 bp) insertion, or “GT” (2 bp) deletion. Additional nicking sgRNA targets were designed for PE3. Listed sequences indicate the “CTT” or “Loxp” insertion or “GT” deletion (on-target products), potential indels at the pegRNA nicking site (indel 1), and potential indels at the nicking sgRNA targeting site (indel 2). On target, Indel 1 and Indel 2 frequencies were analyzed in b–d. b–d Miseq analysis of editing efficiency at the HEK3 locus for “CTT” insertion (b), “LoxP” insertion (c), and “GT” deletion (d) in H1 hESCs using PE tools with/without p53DD, as well as the potential indels frequency. For insertion: n = 3 independent electroporation reactions for PE2 and PE3, without p53DD conditions. n = 2 independent electroporation reactions for PE2 and PE3, with p53DD conditions. For deletion: n = 3 independent electroporation reactions for each condition. Values presented as mean ± S.D. p values were calculated by ordinary one-way ANOVA. e. Miseq analysis of editing efficiency for introducing SERPING1 (c.351delC) in H1 hESCs using PE tools with/without p53DD. n = 2 independent electroporation reactions for PE3 with p53DD conditions. n = 3 independent electroporation reactions for all other conditions. Values presented as mean ± S.D. p values were calculated by ordinary one-way ANOVA. The source data of b–e are provided in Source Data file.

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