Use the CRISPR/Cas9 system in vitro to cut DNA in a tube and analyze the results with gel electrophoresis.

• 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

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.

IMPORTANT Storage notes: The CHOPPED! reagents will be shipped overnight on ice and will need to be stored in the freezer immediately upon arrival. We strongly recommend storing the reagents in the coldest part of the freezer where temperature fluctuations are minimal (e.g., in the back of the freezer instead of the freezer door). The Cas9 Nuclease has greater stability when stored at higher concentrations. For this reason, you will need to dilute the enzyme shortly before use. See the Instructor’s Guide for more details. Reagents are stable for 12 months when stored according to these recommendations.

Required equipment:
- Micropipettes and tips
- 37 °C heat source (e.g., incubator, water bath, or heat block)
- Gel electrophoresis and fluorescence visualization system
- Optional: Vortex and microcentrifuge