Generation of SH-SY5Y HTT knockout cell lines by CRISPR-Cas9

Four guidance RNA (gRNA) sequences targeting the human huntingtin gene (HTT, Gene ID 3064) with minimal off target effects were designed using the online Synthego CRISPR design tool (https://design.synthego.com/#). The four gRNAs are: gRNA1: HTT + 3116206-ACAAGCCUGAAAGGCAGCUU; gRNA2: HTT + 3116154-GCUGCUCACCCUGAGGUAUU; gRNA3: HTT + 3116100-AGGCUUACUCGUUCCUGUCG and gRNA4: HTT-3116085-GACAGGAACGAGUAAGCCUG. gRNAs were dissolved in nuclease-free Tris–EDTA (TE) buffer at a final concertation of 30 μM. Streptococcus pyogenes Cas9 protein with two nuclear localization signals (Cas9 2NLS) was obtained from Synthego and used at a final concentration of 20 μM. sgRNA and Cas9 2NLS at a molar ratio of 9:1 were used to form ribonucleoprotein complex at room temperature, which was then delivered to SH-SY5Y cells using the Neon electroporation system (1100 V, 50 ms width, 1 pulse). Forty-eight hours after electroporation, aliquots of cells were first analyzed for cleavage efficiency by mismatch cleavage assay as described below. Based on the calculated cleavage efficiency, cells transfected with gRNA2 were further serial-diluted to obtain single cell colony in 96-well plates. After colony expansion, colonies were first screened for Htt expression levels by Dot Blot (Additional file 1: Figure S1) and positive clones were selected for further validation by Sanger sequencing.