Journal: Current protocols in molecular biology

Article Title: Generating Single Cell-Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9.

doi: 10.1002/cpmb.100

Figure Lengend Snippet: Figure 1 Schematic outline of the knockout process. The procedure includes (1) choosing a knockout strategy; (2) selecting gRNA target sites and performing vector cloning (Support Pro- tocols 1–3, Basic Protocol 1); (3) introducing CRISPR plasmids by transfection or transduction (Basic Protocols 2–5); (4) isolation and expansion of single-cell clones (Basic Protocol 6, Alternate Protocol 1); and (5) knockout verification by western blot analysis, PCR, and/or Sanger sequencing (Support Protocol 4, Basic Protocols 7–9).

Article Snippet: HEK293T cells (ATCC CRL-3216) CRISPR plasmid DNA (“all-in-one” plasmid or gRNA/Cas9 plasmids from Basic Protocol 1) psPAX2.0 plasmid (Addgene plasmid no. 12260) VSVG plasmid (Addgene plasmid no. 12259) 2 M CaCl2 Solution B (2× HEPES-buffered saline; see recipe).

Techniques: Knock-Out, Plasmid Preparation, Cloning, CRISPR, Transfection, Transduction, Isolation, Clone Assay, Western Blot, Sequencing