a) Schematic representation of cloning strategy. SNCA gene map showing exons (1a and 1b non-coding, 2–6 coding) and the 3′UTR. Transfection of sgRNA targeting the 3′end of exon 6 induces a DSB near the stop codon (TAA). Donor vector design contains 5′ homology arm of 790 bp encompassing part of intron 5 and exon 6 upstream from the stop codon and the NanoLuc-3′ homology arm of 800 bp downstream of the stop codon containing part of the 3′UTR. Co-transfection of donor vector with the CRISPR/Cas9 construct precisely incorporated the NanoLuc right before the stop codon by HDR of the SNCA gene. ( b) Following puromycin selection and single cell dilution, genomic DNA from all surviving isogenic colonies were screened for the NanoLuc insert with pNL1.1 NanoLuc vector and HEK293T LVX cells as controls. From 15 colonies recovered, two were positive for the NanoLuc insertion. ( c) Gene specific PCR with primers in the intron 5 (A) and the 3′UTR of SNCA showed colony 9 had a heterozygous insertion in 293T-SNCA-3′NL cells (Lane 1); PCR with forward primer on the NanoLuc (B or NanoLuc Internal Forward Primer) and the same 3′UTR reverse primer (cDNA sequencing Reverse Primer) showed comparable amplification of the NanoLuc tagged allele (Lane 2); PCR of the wild-type α-SYN and NanoLuc from the HEK293T LVX as controls (Insertion Confirmation Forward Primer and cDNA sequencing Reverse Primer) (Lanes 3 and 4) ( d) Excerpt of Sanger sequencing results showing insertion of the NanoLuc sequence with restriction sites precisely before the stop codon of SNCA and with correct continuation of the 3′UTR after the NanoLuc sequence.