CRISPR editing Flashcards
What does CRISPR stand for?
Clustered regularly interspaced short palindromic repeats.
How was CRISPR gene editing discovered?
A repeated sequence downstream of the translation termination codon for the iap gene was discovered. Then Cas enzymes were discovered. Human genome project reveals salient points. Elucidated molecular mechanisms of Cas endonuclease.
How does CRISPR-Cas mediated prokaryotic immunity work?
- CRISPR adaptation (Cas1/Cas2-mediated) Excision of short 32 nt sequence complementary to invader which is integrated into the host genome as a spacer. 2. Expression of CRISPR loci and processing to produce CRISPR(cr)RNA. 3. Interference i.e. mobile genetic element (MGE) silencing mediated by Cas nucleases.
How are Cas1/2 conserved?
They are conserved. It’s at the expression and interference stages that the class/types begin to differ. Hosts can have more than one CRISPR system that work in tandem.
What does type II CRISPR-Cas cleavage require?
This system requires transactivating (tracr)RNA as well as crRNA to make a guide RNA for targeting of invader by Cas9 (single protein).
How do hosts prevent self-targeting of gDNA by Cas effectors?
Mobile genetic element from target. Protospacer adjacent motif PAM for Cas9. Cas9 mediated cleavage can only proceed when PAM is present i.e., Cas9 won’t cleave host gDNA.
What’s the relevance of bacterial Cas9-mediated adaptive immunity to gene editing?
Experiments in vitro by Doudna revealed that a single chimera guide RNA can be used to direct an endonuclease (Cas9) to induce a DNA double strand break.
What are the steps to complete gene editing in the lab?
Design, cloning and testing of Cas9 guides, nucleofect iPSC, selection pressure, isolating single cell clones, PCR-based screening, scale-up QC.
Why CRISPR-Cas editing?
Bio-engineering complexity, customisation, time, cost.
What were the original ways to edit DNA?
Zinc finger nucleases, transcription activator like endonucleases.
What are the applications of CRISPR-Cas editing in basic science?
Understanding neurodegeneration.
What are the current limitations of CRISPR-Dx?
Required a simplification of chemistries (often multi step), equipment, extraction of nucleic acids.
What are CRISPR-Cas therapies for human disease?
Sickle cell disease, cancer, development of disease resistant parasites.
What are the current limitations of CRISPR?
Editing dictated by presence of a PAM nuclear target sequence (re engineered nucleases)
Delivery (smaller Cas9 derivatives);
Immunogenicity (Cas9 isolated from bacteria that cause infection) Off target effects/safety.
