Extended Data Fig. 2: FAN1 knockout and D960A editing using CRISPR-Cas9. | Nature Neuroscience

Extended Data Fig. 2: FAN1 knockout and D960A editing using CRISPR-Cas9.

From: Exome sequencing of individuals with Huntington’s disease implicates FAN1 nuclease activity in slowing CAG expansion and disease onset

Extended Data Fig. 2

a, Schematic depicting CRISPR-Cas9 targeting of exon 2 of FAN1 in Q109-n1 and Q109-n5 using two guide RNAs (gRNAs) to induce a 94 bp deletion leading to a premature stop codon and FAN1 knockout. The primer pair used for PCR screening of exon 2 after CRISPR is also shown. b, Diagnostic PCR screen using primers FAN1-KO-F and FAN1-KO-R showing representative banding patterns for iPSC lines with the three possible FAN1 genotypes after CRISPR: FAN1+/+ (230/230 bp; wild-type), FAN1+/− (230/135 bp) and FAN1−/− (135/135 bp). c, Sanger sequencing of PCR products demonstrates the targeted 94 bp deletion in exon 2 of FAN1. d, Undifferentiated iPSCs stained for the pluripotency marker OCT4. iPSC-derived neurons stained positive for the neuronal marker MAP2 (red) and CTIP2 (green). All nuclei are counterstained with DAPI (blue). e, Schematic depicting CRISPR-Cas9 targeting of exon 13 of FAN1 in Q109-n5 using a homology directed repair (HDR) template. A single guide RNA sequence (grey) and a 122 bp HDR template containing the desired gene edit coding for an amino acid change (D960A) were utilised to generate FAN1-nuclease dead clones. The HDR template contained two silent mutations (lowercase) to prevent Cas9 re-cutting of the edited region and to introduce a StuI restriction site for diagnostic screening. f, Restriction digest with StuI confirms Q109-n5 FAN1+/+, Q109-n5 FAN1+/D960A and Q109-n5 FAN1D960A/D960A genotypes. StuI cleaves the 442 bp PCR product into 124 and 318 bp products only in the presence of the silent 2868 G > A mutation. The parental Q109-n5 line is also shown as a negative control (right). g, Sanger sequencing of PCR products confirms successful introduction of D960A variant. h, Virtual karyotyping of iPSC lines using copy number variant (CNV) analysis at the time of repeat expansion experiments. CNV analysis reveals small deletions at 2q22.1 and 14q24.3 and duplications at 12q14.2 in all samples. Duplication of chromosome 1 shown in all but one sample.

Source data

Back to article page