CRISPR Targeting

Guide RNA Design

gRNA sequence (20 bp): Random gRNA

Off-Target Tolerance

Maximum mismatches: 3 Show off-target sites

DSB Repair Pathway

Repair mechanism: NHEJ (error-prone) HDR (template-directed)

Target Sites

Off-Target Sites

What is CRISPR-Cas9?

CRISPR-Cas9 is a genome editing tool that uses a guide RNA (gRNA) to direct the Cas9 nuclease to a specific DNA sequence. The gRNA must match the target and be adjacent to a PAM sequence (NGG for SpCas9).

On-target: Perfect match to gRNA + PAM → Cas9 creates double-strand break (DSB)

Off-target: Sequences with mismatches can still be cleaved, especially if mismatches are PAM-distal (far from PAM). Off-target scoring uses position-weighted penalties.

NHEJ: Non-homologous end joining is error-prone, creating random insertions/deletions (indels). Often causes frameshifts → gene knockout.

HDR: Homology-directed repair uses a donor template for precise edits, but is less efficient than NHEJ and requires template homology.