Extended Data Fig. 6: Off target sites validation in HEK293T cells.
From: Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag
a, Indel rates for nucleotide substitution, targeted 1-bp deletion, and 1-bp insertion pegRNAs with PE2 at 8 potential off-target sites of top 20 OTs identified by GUIDE-seq that overlap with in vitro PE-tag for HEK4 locus pegRNA in HEK293T cells. Frequencies of editing were quantified by deep sequencing from PCR amplicons spanning each locus. Results were obtained from three independent experiments and presented as mean ± SD. *P < 0.05, ** P < 0.01 and *** P < 0.001 by two-way ANOVA with Tukey’s multiple comparisons test. b, Indel rates for nucleotide substitution, targeted 1-bp deletion, and 1-bp insertion pegRNAs with PE2 at 12 potential off-target sites of top 20 OTs identified by GUIDE-seq but absent in the in vitro PE-tag for HEK4 locus pegRNA in HEK293T cells. pegRNA sequence composition and type of sequence modification encoded is indicated in the legend, where the terminal numbers indicate the different HA lengths within the RTT. Frequencies of editing were quantified by deep sequencing from PCR amplicons spanning each locus. Results were obtained from three independent experiments and presented as mean ± SD. *P < 0.05, ** P < 0.01 and *** P < 0.001 by two-way ANOVA with Tukey’s multiple comparisons test. c, Editing outcomes with PE2 and 1-bp deletion pegRNA at HEK4 MISS-2 and MISS-7 in HEK293T cells. Frequencies of editing were quantified by deep sequencing of PCR amplicons spanning the locus. CRISPResso output shown for sequencing data.
