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. 2021 Jul 9;12:4219. doi: 10.1038/s41467-021-24454-5

Fig. 3. Genome editing efficiency and specificity of SluCas9 and sRGNs at endogenous loci in mammalian cells.

Fig. 3

a The effect of guide length on sRGN3.1 editing efficiency. The HBB_R01_T2 site was targeted by sgRNAs of lengths from 15 to 25nts (guide_80-90) using RNP nucleofection and two different RNP concentrations. The editing efficiency was quantified by ddPCR; data are presented as mean ± SEM with n = 3 independent biological replicates. b Guide concentration dose-response. SluCas9 and sRGN3.1 activity after RNP nucleofection at the HBB_R01 locus (guide_87) in HEK293T cells. Curve fitting was conducted by the least-squares methods, r2 = 0.92 (sRGN3.1) and 0.98 (SluCas9), the editing efficiency was measured by ddPCR and normalized to the maximum for each nuclease, data are presented as n = 2 independent biological replicates. EC50s were 5.7 pmol for sRGN3.1 and 12.1 pmol for Slu. c The median activity on 24 genome targets. Comparison of genome editing efficiency at endogenous targets in HEK293T cells following RNP nucleofection. Twenty-four different guides (5′-NGGG-3′ PAM), across seven genomic loci (VEGFA, HBB, FANCF, Apolipoprotein (APP), USH2A, and EMX), were assessed by amplicon sequencing (22 nt guides for SluCas9 and sRGN3.1: guides_32-55; 20 nt guides for Spy: guides_56-79, Supplementary Table 1). SpyCas9, SluCas9, and sRGN3.1 without guide RNA served as controls and had a median editing of 1.08 ± 0.64%; data are presented as n = 3 independent biological replicates. The right data from the left panel with average editing on each target normalized to SpyCas9. Red line = mean. The significance was determined using the one-sample Wilcoxon signed-rank test against a theoretical median of one with an alpha of 0.05, ns = not significant, (*) = p ≤ 0.05, SpyCa9s vs SluCas9 p = 0.68, SpyCas9 vs sRGN3.1 p = 0.05. d GUIDE-Seq analysis for SpyCas9, SluCas9, and sRGN3.1 on HBB_R01 and VEGFA_T2. On-target sequences in 5′–3′ orientation are shown in the top row of each panel. Identified off-targets are listed in the subsequent rows and ranked by the number of reads. Matches to the on-target are shown as (.), mismatches are highlighted. Note that for the SpyCas9 HBB_R01 off-target analysis, only 47 of the 82 detected off-targets are shown on distinct rows, because 37 of the determined off-target loci are distributed at two identical off-target sequences (AGgaacAtggatgaagCtgg was found 27 times, AGgaacAtggatgGagttgg 10 times). These duplicates were combined into the respective two rows. The full off-target list for each sample can be found in the source data file. Data are presented as n = 1 for each guide.