CRISPR-Cas9 Gene Editing

CRISPR-Cas9 Gene Editing 3D lets students operate the molecular scissors revolutionising modern genetics and biotechnology. A translucent bilobed Cas9 protein carrying a glowing pink 20-nucleotide guide RNA (gRNA) slides along a 3D double-stranded DNA helix. When students drag the Cas9 complex onto the golden 3-base PAM motif (5'-NGG-3'), the target DNA unwinds into an R-loop bubble and the HNH and RuvC nuclease domains make a double-strand break. Students can switch between Uncut, NHEJ Gene Knockout (indel frameshift), and HDR Precise Edit (donor template knock-in) and verify the resulting allele sizes on a 3D agarose gel electrophoresis slab.

• Drag the cyan Cas9-gRNA complex along the DNA double helix until it locks onto the golden 5'-NGG-3' PAM motif near the centre.
• Observe how the DNA strands unwind into an R-loop bubble inside Cas9 when the 20-nucleotide guide RNA matches the target sequence.
• Switch Repair pathway between NHEJ Gene Knockout and HDR Precise Edit to see red indel disruption versus green donor sequence integration at the cut site.
• Watch the three lanes on the 3D Agarose Gel Electrophoresis slab migrate at different speeds for wild-type, NHEJ deletion, and HDR knock-in alleles.

1. 1Explain the respective roles of the Cas9 endonuclease protein, the 20-nt single guide RNA (gRNA), and the 5'-NGG-3' PAM sequence.
2. 2Describe how Cas9 unwinds target DNA into an R-loop and creates a blunt double-strand break three base pairs upstream of the PAM.
3. 3Compare error-prone Non-Homologous End Joining (NHEJ) for gene knockout with Homology-Directed Repair (HDR) for precise sequence editing.
4. 4Interpret gel electrophoresis band migration to distinguish edited alleles from wild-type DNA.

Questions teachers ask