Explain how the CRISPR-Cas9 system can be manipulated to edit genes for research and gene therapy.
Tetrapods Example: Snakes
Snakes are tetrapods, supported by fossil evidence (Tetrapodophis amplectus) and developmental homologies (limb buds in snake embryos).
Limb development is controlled by Sonic Hedgehog (Shh) across tetrapods.
Reading Gene Diagrams
A: +1 TSS (Transcription Start Site)
C: Distal regulatory element where transcription factor ZRS binds (ZRS promotes Shh expression, so it's an activator).
B: Indicates 1 million bases between A and C.
Sonic Hedge Hog (Shh)
Morphogen: substance with non-uniform distribution controlling development. Different parts of the limb form based on how much Shh it receives.
Signaling Molecule: In the absence of Shh, PTCH receptor inhibits SMO, which inhibits SUFU, which inhibits Gli1. When Shh binds to PTCH, PTCH is inhibited via protein shape change, activating SMO (signal transduction).
Shh and Cancer
Many normal developmental pathways like Shh signaling are mutated in various cancers.
PTCH's normal function is to inhibit proliferation; loss of function mutations in PTCH are found in basal cell carcinomas, indicating it is a tumor suppressor.
Shh in Snake Development
Snakes have a ZRS enhancer deletion, resulting in loss of proliferation in limb buds.
CRISPR-Cas9 System
Derived from bacteria's defense against phages.
Bacteria cut up phage DNA using Cas9 and guide RNA.
Guide RNA genes controlled by a promoter in the bacteria genome.
Cas9 + guide RNA complex: guide RNA binds to virus DNA target sequence, Cas9 nuclease cuts backbone, creating a sequence-specific double-strand break.
CRISPR Immunity
Viral DNA integrated into bacteria genome is transcribed to produce guide RNAs matching previous viruses. This is an adaptive immune system.
Gene Editing
Scientists use CRISPR-Cas9 for gene editing by introducing a guide RNA complementary to a target gene and Cas9 into cells, causing a double-strand break.
DNA Repair: Integration of a new sequence uses host cell DNA Damage Response to repair the break using a provided template.
Gene Therapy
Deliver targeted nucleases to cells by physical, chemical, or viral methods.
Ex vivo: Extract stem or progenitor cells, modify them, and reintroduce them to the patient.
In vivo: Direct delivery to patient using viral (e.g., AAV, Lentivirus) or non-viral (e.g., Lipid nanoparticle) delivery vehicles.