2 Neuroscience2.9 Genetics
2.9.7 Gene modification and editing, including CRISPR Cas and similar tools
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Written by Resident Doctor Committee · Published
Site-specific nucleases
Site-specific nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Cas proteins, cut both strands of DNA at a chosen locus, and the cell's repair pathways then make the edit.
Cas9 is aimed by a guide RNA (gRNA), whose roughly 20-base targeting region pairs with the target by complementary bases.
ZFNs and TALENs are aimed by engineered proteins that must be redesigned for each target. Cas9 is retargeted just by changing the gRNA, so one nuclease can be aimed at many loci at once.
Repair after a Cas9 double-strand break
Feature | Non-homologous end joining | Homology-directed repair |
|---|---|---|
Template | None | Supplied donor with homology arms |
Accuracy | Error prone, leaving small insertions or deletions (indels) | Precise copy of the designed change |
Cell cycle | Most cells, including non-dividing ones | S and G2 phases only |
Typical use | Gene knockout through loss of function | Correcting a variant or inserting sequence |
Mature neurones support little conventional homology-directed repair.
Cas9 variants that do not cut both strands
Catalytically dead Cas9 (dCas9) binds its target without cutting and blocks transcription by steric hindrance. This is CRISPR interference, and it leaves the sequence intact.
Nickases cut one strand only.
Uses in psychiatric research
Human induced pluripotent stem cells (hiPSCs) are adult somatic cells, such as fibroblasts, that transcription factors have reprogrammed to a pluripotent state resembling embryonic stem cells.
Introducing or correcting a risk variant in hiPSCs gives closely matched lines, so genetic background does not confound the comparison.
Off-target edits at similar sites are one technical limit. Incomplete editing is another, because it leaves cells with mixed genotypes.