Px458 crispr cas9 vector

The 5′-AGGAGCTGGCGAGCGCCCTGAGG-3′ nucleotide sequences in exon 1 of the mouse Hoip gene were selected as the targets. Double-stranded target DNAs were prepared by annealing synthesized oligo DNAs, and then incorporated into the BbsI site of the px458 CRISPR/Cas9 vector (Addgene, Watertown, MA, USA). Each plasmid was transiently introduced into Neuro2a cells, and 2 days after transfection, the GFP positive cells were sorted by a FACSAria IIIu (BD Biosciences, San Jose, CA, USA). Single clones were obtained by limiting dilution. Genome editing of the Hoip gene was confirmed by genome sequencing and immunoblotting.

The first coding exon of Nestin was selected for gRNA design. CRISPRs were designed at http://crispr.mit.edu, as provided by the Zhang laboratory and cloned into the pX458 CRISPR/Cas9 vector (Addgene). The sequences used to clone the G1, G2, and G3 gRNAs were presented in Supplementary Figure 2. Two days after H1299 cells were transfected with control (Cas9-GFP) and Nestin-knockout (G2-Cas9-GFP) plasmids, those expressing GFP were sorted using an Influx Cell Sorter (BD, USA) and cultured on 12-well plates. Twenty-four hours later, half of the Nestin-knockout cells were transfected with GFP-lamin A/C. The cells were then cultured for 3 days and harvested for further experiments.