Use the CRISPR/Cas9 system in vitroto cut DNA in a tube and analyze the results with gel electrophoresis. Investigate how the Cas9 nuclease can be programmed to target virtually any DNA sequence—just by changing the guide RNA.  The introduction of CRISPR/Cas into the biotechnology toolkit has revolutionized gene targeting and gene editing. In this lab, students will use the CRISPR/Cas system in vitro (in a test tube) to analyze the function of its molecular components. They will use two different guide RNAs to direct Cas9 nuclease to cut a DNA sample at two different locations and then analyze their resulting DNA fragments using gel electrophoresis. This gene targeting lab will allow students to investigate how the Cas9 nuclease can be programmed to target virtually any DNA sequence—just by changing the guide RNA.  This activity will demonstrate how the CRISPR/Cas system is both programmable and specific and why it is such a powerful genome editing tool.  Techniques: Micropipetting, gene targeting, gel electrophoresis Topics: Genome editing, biotechnology, CRISPR/Cas9 Time required: Two 45-min class periods or one 90-min block Level: General high school through college