How do you design sgRNA sequences for CRISPR-Cas9?

The sgRNA is responsible for the specificity of the CRISPR-Cas9 system, which consists of two parts, a constant region and a variable region. The constant region, also known as the tracrRNA, is the scaffold sequence that binds to Cas9 protein. The variable part is the around-20-nucleotide-long crRNA, which is complementary to the target gene and determines the specificity of the sgRNA.

To design a sgRNA that is specific to target DNA, several design criteria must be followed:

• The GC content of the sgRNA should be in range of 40-80%.
• The length of the crRNA is 17-24 base pairs.
• The protospacer adjacent motif (PAM) sequence should not be included in the sgRNA.
• The target sequence can be on either DNA strand.

Several online tools (e.g., CRISPR Design or CHOPCHOP) can help to detect the PAM and design the crRNA sequence. The general steps these tools follow to design a sgRNA sequence are:

• Identify the PAM sequence in the target DNA sequence. For Cas9 nuclease of Streptococcus pyogenes, the PAM sequence is NGG.
• Determine the 5’ start of the sgRNA sequence by counting around 20 nucleotides upstream of the PAM sequence.
• Determine the sgRNA sequence.