Figure 1

Our dual gRNAs remove the C9ORF72 repeat site in human cell. (A) The C9ORF72 genome structure (GRCh37/hg19) and our dual-gRNA design (gRNA1-2 pair or gRNA 3-b pair) for removal of the C9ORF72 repeat site. The gRNAs described here are SpCas9-based. The primers flanking the gRNA editing sites labeled as red arrows used for genotyping the removal. The primers in last exon (exon 11, Ex11) labeled as black arrows used for detecting C9ORF72. (B) All available gRNAs lie between the repeat site and exon 1b (Ex1b). The distance between the repeat site and exon 1b is 35bps. The gRNAb is in antisense orientation, and the rest of gRNAs are in sense orientation. (C) Off-target summary of gRNAs we examined by using an online gRNA design tool (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design). The off-target details are shown in Supplementary Table 1. (D) Genomic DNA PCR for detecting the removal of repeat site by gRNA1-2 and gRNA3-b. The HEK293 cells constitutively expressing Cas9 were infected with the lentiviral control, gRNA1-2, and gRNA3-b, respectively (also see Supplementary Fig. 2). The repeat site deletion (ΔRepeats) or repeat site plus exon1b (E1b) deletion (ΔRepeats + E1b) appeared in cells infected with gRNA1-2 or gRNA3-b. Due to the high GC content of the DNA region we amplified, PCR amplicons containing the repeat site and its neighboring sequences (labelled as asterisk) did not appear in control group in the PCR condition with low amount of high GC buffer. High GC buffer condition: from red to yellow, usage of high GC buffer from high to low %. The amplicons of C9ORF72 last exon were used for PCR reaction control. The primers used here were illustrated in (A). (E and F) The repeat deletion bands (Δ) shown in (D) were applied for both forward and reverse Sanger sequencing. The editing site fusions took place upstream of the PAM (the protospacer adjacent motif, NGG) sites.