CRISPR is the coolest technology in a biologist’s toolbox because of its unique blend of being extremely powerful yet relatively simple to use. The technique relies on two basic components: a Cas nuclease that cuts the DNA and a guide RNA that tells the nuclease precisely where in the genome to cut.

The best way to approach any challenge is to break it down into smaller steps. Broadly speaking, every CRISPR experiment can be divided into three main steps:

1. Design - Ensure that you select the optimum guide RNA and other components for your experiment

2. Edit - Introduce the CRISPR components into cells to allow the genome engineering to occur

3. Analyze - Verify the effectiveness of your experiment and move on to the next steps

Step 1: Design the CRISPR sgRNA

The first step in your CRISPR experiment is to design the customizable guide RNA to target your DNA sequence. The specificity of the guide RNA will greatly influence the success of your CRISPR experiment, as any unintentional binding to random sites could have detrimental effects on the cell.

Step 2: Edit DNA Precisely with CRISPR

Once the sgRNA is designed and synthesized, you can precisely edit the desired DNA sequence in any genome... right after you have chosen the appropriate CRISPR nuclease. Gone are the days when CRISPR experiments automatically meant using the Cas9 nuclease. Now, depending on the application and specific experimental needs, a variety of alternative nucleases exist. Plus, you need to ensure that the sequence you want to edit contains the PAM sequence that the nuclease absolutely needs to cut. Last but not least, after all CRISPR components have been selected, one more choice remains. What is the best delivery system to introduce the CRISPR components into the cell?

Step 3: Analyze Data from CRISPR Experiment

The last step in the CRISPR workflow is to analyze the editing efficiency and to determine the DNA sequences at your target region after being edited. You picked the best sgRNA and used an optimum transfection method, and now you need to review the quality of your editing and make plans for what comes next! Choosing an accurate analysis method and using it efficiently are essential factors influencing the success of this step.