Fig. 2: Tag-seq global profiles and molecular characterizes off-target cleavages induced by Cas-nuclease. | Communications Biology

Fig. 2: Tag-seq global profiles and molecular characterizes off-target cleavages induced by Cas-nuclease.

From: Tag-seq: a convenient and scalable method for genome-wide specificity assessment of CRISPR/Cas nucleases

Fig. 2

a–c PEI-based transfection and electroporation (Lonza Nucleofection)-based methods displayed comparable results in detection of off-target sites at EMX1, PD1, and CLTA4 loci. d, e Off-target sites detection with Tag-seq. SpCas9 and AsCpf1 targeting EMX1 and Site 6, respectively, induced off-target cleavages in HEK293T and MCF7 cells. For visualization, the targeted sgRNA was shown in the first line and the on-target and the off-target sites were shown without or with mismatches to the sgRNA sequence by color highlighting. Sequencing read counts were shown to the right of each site. Due to the limited space, the off-target sites for Site 6 were not completed displayed, and a full list was displayed in Supplementary Fig. 3. f–i Tag-seq displayed the characteristics of the SpCas9/Cpf1-induced-DSBs. Cutting at 3-4 bp upstream from the NGG PAM site and resulting in 1-bp overhangs at EMX1 on-target (f) and off-target site 1 (OT-1) (g) sites for SpCas9 in both HEK293T and MCF7 cells. Resulting multiple overhangs at Site 6 on-target (h) and off-target site 1 (OT-1) (i) sites for AsCpf1 in both HEK293T and MCF7 cells. Red dotted lines indicated the cutting sites.

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