Design a CRISPR-Cas experiment

When designing a CRISPR-Cas experiment, several factors need to be considered:

• Select the desired genetic manipulation: This will allow you to choose between different CRISPR-Cas components. CRISPR-Cas is normally used for knocking-out cells but it can also be used for editing, repressing or interfering, or activating genes as well.

• Select the target sequence and design the gRNA:

  • Know the cell line or organism and the genomic sequence: Since sequence variation between gRNA targeting sequence and target DNA leads to reduced yield, sequencing the region to edit is highly recommended.

  • Select gene and genetic element to manipulate: Depending on the goal of the experiment, the DNA target region can be part of a promoter, an expressed exon, or sequences that relates to specific domains of the protein.

  • Design the gRNA sequence: Make sure that the PAM sequence for the selected Cas protein is present in the target DNA, otherwise there will be no cleavage. Since the PAM sequence is essential for Cas to bind the target DNA, avoid including it in the gRNA or the experiment will fail too. Another important aspect for designing the gRNA is the on-target and off-target activity of the gRNA. Try to design a gRNA sequence with a high on-target activity and very-low off-target activity. Otherwise, different target DNAs might be cleavaged or the efficiency might be too low. In order to help with this process, there are many online tools to help researchers selecting the optimal gRNA sequence.

  • Synthesize and clone desired gRNAs: The designed gRNA can be directly ordered already cloned into a plasmid or it can be cloned in the laboratory.

• Deliver Cas9 and gRNA: It is important to choose a delivery method to introduce the CRISPR components that is compatible with the experimental system. The efficiency of the experiment may vary depending on the selected method. Therefore, it is recommended to optimize the delivery conditions beforehand.