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. 2021 Jun 23;4:771. doi: 10.1038/s42003-021-02304-w

Fig. 2. CAG repeat contraction in R6/2 ES cells.

Fig. 2

a Procedure for establishing ES cells from R6/2 mice. ES cell stemness was examined by Oct3/4 immunostaining and ALP activity. b Design of gRNAs and primers for genotyping (Fw and Rv). The orange background color shows the area of the repeat tract. Bold characters with a red box in the DNA sequence indicate the second G in NGN-PAMs. c PCR-based screening for successfully contracted clones. The red arrows indicate the original size of the PCR amplicon. d Direct sequencing of PCR amplicon. Clone #11 in gRNA-S2/SpCas9-NG are shown as an example. As we amplified human HTT transgene from mouse ES cells, the overlapping electrogram at the 3’ is due to a variety of repeat length or/and errors during PCR and sanger sequencing. e Summary of PCR-based screening and direct sequencing. f Violin plot for the number of CAG repeats in ES cell clones transfected with gRNA-S1/SpCas9-NG or gRNA-S2/SpCas9-NG expression vector. Black triangles indicate clones without detectable band-shift. Red diamonds and blue dots indicate clones with downward band-shift; red diamond: in-frame CAG repeat sequence; blue dot: out-of-frame CAG repeat sequence.