CRISPR-Cas9 (lect 6 and 7) Flashcards
What are the components of the CRISPR-Cas9 system?
-guide RNA (synthetic RNA made up of spacer and scaffold)
-CRISPR-associated endonuclease (Cas9)
How do mammalian cells repair Cas9-induced breaks?
-non-homologous end joining (joins broken ends together, error-prone)
-homology directed repair (uses homologous donor DNA to repair)
How can genome edits be validated?
-mismatch cleavage assay (quickly screen population to see if edit was successful, should detect presence of gRNA and Cas9)
-sequencing (Sanger trace will be disrupted compared to WT)
What is homology-directed repair used for experimentally?
-gene corrections
-knock-ins
-tagging proteins
How can homology-directed repair be made more efficient?
-suppress NHEJ machinery (so will use HDR instead!) (eg. degrade DNA ligase IV)
-synchronise cells to be at cell phases where HDR more efficient than NHEJ
-use small molecule enhancers
-use GFP-tagged Cas9 (allows easy sorting of cells which have successfully been transfused)
What occurs in DNA cleavage in the CRISPR-Cas pathway?
3 bases after PAM site, the DNA is cleaved, creating a blunt-ended double strand break
-co-ordinated by HNH (hydrolyses scissile phosphates of target strand) and RuvC nucleases (hydrolyses scissile phosphates of non-target strand)
DNA cleavage by HNH nuclease
Cleaves scissile phosphate of target strand
-1 metal ion hydrolysis
-3 catalytic active site residues co-ordinate Mg2+
DNA cleavage by RuvC nuclease
Cleaves scissile phosphate of non-target strand
-2 metal ion hydrolysis
-4 catalytic active site residues co-ordinate 2 metal ions and activate H2O for nucleophilic attack
What is catalytically dead Cas9 (dCas9)?
Cas9 with double mutation (point mutations in HNH and RuvC domain) meaning it is no longer catalytically active (can’t cleave either strand but can still bind to them!)
-versatile tool -can target DNA, which is useful for activating/repressing genes
How can Cas9 be engineered to created an overhanding ds break?
-mutating Cas9 so only one of the nucleases (HNH or RuvC) is catalytically inactive -> nickase! - will create a ss break
-using two nickases will create ss breaks on each strand (therefore, a ds break!)
-useful because ss breaks are more efficiently repaired than ds breaks so will have less off-target effects!
How can dCas9 be used for gene regulation?
-CRISPR activation (fuse dCas9 to a tf to activate gene expression)
-CRISPR interference (fuse dCas9 to a transcriptional repressor to downregulate gene expression)
How can dCas9 be used experimentally?
For gene regulation
-CRISPR activation
-CRISPR interference
For epigenome editing
For base editing
-base editing
-prime editing
How can dCas9 be used for epigenome editing?
dCas9 fused to acetyl transferase domain of p300, allowing it to be recruited to specific promotors/enhancers
How can Cas9 be used for base editing?
Targets a locus and irreversibly converts that bp into another bp
-no need for ds breaks, HDR or donor DNA templates!
Base editing approach:
-targets one of the nts of the ss non-target strand accesible outside of Cas:sgRNA:DNA complex
Prime editing approach:
-nickase Cas9 fused to engineered reverse transcriptase
-sgRNA extended, to encode gRNA and repair template (will include mutated sequence!)
Example of C to T conversion using base editing with Cas9
-converts C to U on accessible ss non-target DNA strand by cytidine deaminase (fused to Cas9)
-mutated nickase Cas9 (inactive RuvC domain) cleaves non-edited DNA strand
-cellular DNA repair replaces strand containing G, using deaminated (U) strand (with U:A bp) as template so that a T is synthesised instead of a U
