Px330 u6 chimeric bb cbh hspcas9 px330 vector

Guide RNA target sequences within the FUS gene were identified using Feng Zhang lab’s Target Finder (https://zlab.bio/guide-design-resources). Respective forward and reverse oligonucleotides were annealed and cloned into pX330-U6-Chimeric_BB-CBh-hSpCas9 (pX330) vector (Addgene) according to the previously described protocol [13 (link)]. SH-SY5Y human neuroblastoma cells were split onto a 35 mm dish at 50–60% confluency one day prior to transfection. Equal amounts of plasmids (3.6 μg each) carrying upstream and downstream gRNA target sequence (or one plasmid for FUS knockout) were delivered into cells by calcium phosphate transfection. After 24 h, cells were resuspended at ~ 10–20 cells/ml and plated onto 10 cm dishes. Single-cell derived clones were expanded and screened by immunofluorescence and PCR. For sequencing of the edited portion of FUS gene, the PCR product corresponding to the edited allele was cloned into Zero Blunt® TOPO® vector (Life Technologies), and at least four colonies were sequenced. Primers used for PCR screening and TOPO® cloning: ΔNLS lines: 5’-TGGGGACAGAGGTGGCTTTG-3′ and 5’-CCTTCCTGATCGGGACATCG-3′; FUS KO: 5’-ACCATTTGAGAAAGGCACGCT-3′ and 5’-CACGGATTAGGACACTTCCAGT-3′.

Guide RNA (gRNA) sequences targeting NEAT1 gene region that corresponds to the non-overlapping sequence between NEAT1_1 and NEAT1_2 transcripts were identified using Feng Zhang lab's Target Finder (http://crispr.mit.edu/). Two gRNA sequences were selected on each end of the target region to increase editing efficiency. Forward and reverse oligonucleotides Upstream1 5′-TACATCCAAAGTCGTTATGA-3′ and Upstream2 5′-AGAACTGGTATTATCCCAAG-3′; and Downstream1 5′-CCTTGTAAAGGCATAGCCAG-3′ and Downstream2 5′-CAAAACCTGAGTGCGGCCAT-3′ were annealed and cloned into pX330-U6-Chimeric_BB-CBh-hSpCas9 (pX330) vector (Addgene). Plasmids were delivered into SH-SY5Y cells by calcium phosphate transfection. Single-cell derived sub-clones were expanded and screened by PCR using 5′-ATGGGGAAGTAGTCTCGGGT-3′ and 5′-AGGATGAGGGAGGGGATAGC-3′ primers. All primers were custom-synthesized by Merck (Sigma). NEAT1_1 distribution was analysed using Stellaris® FISH Probe Human NEAT1 5′ segment with Quasar^® 570 Dye (Biosearch Technologies, SMF-2036-1).