Fig. 2: The distinctive features of proPE.
a, Amplicon sequencing results of PE3 using tpg/pegRNAs without the tevopreQ1 extension. The ‘eng fraction’ refers to the fraction of transfected engRNA plasmid (see Supplementary Methods), and T and NT indicate targeting and non-targeting tpgRNA, respectively. del, deletion; w/o, without. b, The effect of the amount of engRNA and tpgRNA plasmids was examined at 50% tpgRNA (top) and 1.24% engRNA (bottom) in the total RNA plasmid fraction, respectively. PE2 and proPE2 were used with tpg/pegRNAs without the tevopreQ1 extension. c, PEAR results showing the range of spacer lengths of tpgRNAs for achieving effective proPE editing. nt, nucleotide. PE2 and proPE2 were used for the PEAR-GFP targets, while PE3 and proPE3 were used for the PEAR-mScarlet targets. The tpg/pegRNAs were used without the tevopreQ1 extension. d, PE and proPE editing was assessed in the presence of increasing amounts of degraded RNAs, PBS-less (left plots), (RTT-PBS)-less (right plots), targeting the same site. PE2 and proPE2 were used for the PEAR-GFP target (top plots), while PE3 and proPE3 were used for the PEAR-mScarlet target (bottom plots). The editing values are normalized to the corresponding values from experiments where non-targeting degraded RNAs were co-expressed. The tevopreQ1 extension was used. Lines are fitted to the data points. The statistical significance between the slopes of the fitted lines was assessed using the non-linear module of GraphPad with the two-tailed extra sum-of-squares F-test. The parameters for the linear equations can be found in Source Data Fig. 2. See Supplementary Fig. 1 for further details on d. Each data point in a–c is presented as the mean ± SD of triplicates; in d, data points represent triplicate means normalized to the mean of the non-target control, with the ± SD including error propagation. In b–d, the sample names (PEAR-GFP-C1(S2) and so on) refer to given proPE or PE combinations, the sequences of which are provided in Supplementary Table 6.
