Design Guidelines for CRISPR-Cas9 sgRNAs
In addition to a physical prototype of Hepativir-B, we also designed realistic, prototype CRISPR-Cas9 sgRNAs specific for HBx and for NTCP. For an explanation of how CRISPR-Cas9 works, see the History tab.
The CRISPR-Cas9 system (from S. Pyogenes) uses a customized, single guide RNA (sgRNA) to edit the target gene of interest. The sgRNA has two important regions: the target-specific sequence
(a variable sequence customized to a specific gene which the CRISPR-Cas9 identifies and edits) and the scaffold sequence (a constant sequence that binds Cas9). The Cas9 also recognizes a sequence of 3 nucleotides (e.g. "NGG" where N represents any nucleotide) called the "PAM" sequence which must be adjacent to the target sequence in the gene of interest.
Prototype of NTCP & HBx CRISPR-Cas9
Since NTCP is a gene in the human genome, we used a CRISPR-Cas9 design tool to find sgRNAs to inhibit the gene. Below is one of the main sgRNAs (target-specific sequence in green) identified by the design tool, which could be tested to find the best guide RNA to inhibit NTCP. The image below also shows the “NGG” PAM sequences (marked in blue).
For HBx, the process was much more difficult because the protein is coded in the HBV genome, for which there are few CRISPR design tools. However, we identified four different sgRNAs (target-specific sequences in green) and PAM regions (in blue) which could later be tested to find the best one. One example is below.
