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:
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: