Editing the genome: Mechanisms of CRISPR-Cas9 Flashcards
What are the components of the CRISPR-Cas9 system? (2)
- Guide RNA (gRNA)
- CRISPR associated endonuclease (Cas9)
What is gRNA? (3)
- Also called single guide RNA (sgRNA)
- Synthetic RNA containing a scaffold (Cas9 binding) sequence and spacer (targeting) sequence
- Spacer sequence is user defined, ~20nt and defines the region of the genome to be targeted
How are Cas9-induced DSBs repaired? (2)
- Non-homologous end-joining (NHEJ) or
- Homology-directed repair
What is NHEJ? (2)
- Non-homologous end joining
- Results in insertions/deletions (indels) that are often exploited to create frameshift or knockout mutations
What is HDR? (2)
- Homology-directed repair
- Donor template is used for gene correction of knock-in experiments
How do you validate genome edits? (7)
- Mismatch cleavage/T7E1 assay to select cells with the edit
- Design PCR primers for mutated region and amplify
- Some cells will be completely unedited wildtype, some heterozygous for edit, some homozygous for edit
- Denature and reanneal PCR products, will have a mixed population of unedited and edited products
- T7 endonuclease 1 cleaves mismatched DNA strands so the edited products will show as extra bands because of cleavage
- More cleaved bands = higher efficiency
- Sequencing of PCR products can be used for detection of indels in clonal cell populations
What is TIDE? (2)
- Tracking of indels by decomposition (computational analysis)
- Sanger trace of edited will be disrupted compared to WT
How can you easily identify cells which have integrated the donor sequence?
Encode a restriction site in the repair template so the PCR product can be cleaved to prove integration e.g. NheI
What is the efficiency of HDR?
HDR is very low efficiency (<10% of modified alleles) as NHEJ pathways tend to be favoured over HDR
How can HDR efficiency be improved? (6)
- Make homology arms longer
- Suppression of NHEJ machinery
- Targeted degradation of DNA ligase IV with siRNAs
- Synchronisation of cells at cell cycle stages where HDR is the most active
- Rational design of ssDNA donor template can increase HDR efficiency by up to 60%
- Small molecule enhancers of HDR
How can you sort for successfully transfected cells?
Use GFP-tagged Cas9
How does DNA cleavage occur? (4)
- Occurs 3 bases after the PAM site and produces a blunt-end DSB
- Due to coordinated action of the HNH and RuvC nuclease domains of Cas9
- HNH cleaves the target strand of DNA (complementary to RNA spacer)
- RuvC cleaves the non-target strand
What is the PAM site? (2)
- Protospacer adjacent motif
- Determines where the cleavage occurs in the DNA
What are the nuclease domains of Cas9? (3)
- HNH (target strand)
- RuvC (non-target strand)
- Function independently of each other
How does the HNH domain work? (2)
- Uses a one metal ion mechanism to hydrolyse the scissile phosphates in the target strand backbone
- Active site has 3 catalytic residues
What are the 3 catalytic residues of the catalytic pocket of HNH?
- Asp839
- His840
- Asn863
How does the RuvC domain work? (2)
- Uses a two-metal ion mechanism to hydrolyse the scissile phosphates in the non-target strand backbone
- Active site has 4 catalytic residues
What are the 4 catalytic residues of the catalytic pocket of RuvC?
- Asp10
- Glu76
- His983
- Asp986
What is dCas9?
Catalytically dead Cas9
What are the features of dCas9? (3)
- D10A inactivates the RuvC nuclease so can’t cleave the non-target strand
- H840A inactivates the HNH nuclease so can’t cleave the target strand
- D10A/H840A double mutation produces catalytically dead Cas9 which is one of the most versatile CRISPR tools
Why is dCas9 useful?
Change the catalytic activity but can still target it to specific DNA sequences because of the gRNA
What is Cas9 nickase?
Single mutant of HNH or RuvC domain so only causes a single strand break
What is the main problem with CRISPR-Cas9?
Genome is huge and the spacer sequence is only ~20nt so will likely have homology in multiple areas of the genome resulting in off-target editing
How can you limit CRISPR-Cas9 off-target effects? (3)
- ‘Double nickase’ approach
- Dual gRNAs targeting PAM sites that are 10-20nt apart can target a pair of nickase Cas9s, each cleave one strand so creates a DSB with an overhang
- Reduction in off-target effects due to efficient repair of single strand breaks
What are methods of using dCas9 for gene regulation? (5)
- CRISPRa
- CRISPRi
- SunTag system
- Epigenome editing
- Base editing
What is CRISPRa? (3)
- CRISPR activation
- Fusion of dCas9 with potent transcription factor VP64
- Transcriptional activation of a specific gene without editing
What is CRISPRi? (3)
- CRISPR interference
- Recruitment of dCas9 to promoter in bacteria to block PolII recruitment
- Fusion of dCas9 to a transcriptional repressor such as a KRAB domain can downregulate gene expression
What is the SunTag system? (2)
- Recruitment of multiple copies of a factor e.g. VP64
- Can be used for imaging specific sites of the genome if you attach GFP
What is epigenome editing with dCas9? (3)
- Fuse dCas9 with histone acetyltransferase domain of p300
- Allows recruitment to specific promoters and enhancers
- Increases p300-mediated histone acetylation (H3K27ac) which is transcriptional activator
What is base editing? (3)
- Enables direct, irreversible conversion of one base pair to another at a target genomic locus
- No requirement of DSBs, HDR or donor DNA templates
- Can be used to repair pathogenic SNPs
How does base editing work? (3)
- Base editors target 9nt of the ssDNA non-target strand that remain accessible outside of the Cas9:sgRNA:DNA R-loop complex
- Fused cytidine de-aminases convert C>U specifically on the ssDNA non-target strand
- Target a 5nt window in the ssDNA bubble created by Cas9 on the non-target strand
How does C>T base editing work? (5)
- Most commonly used base editors are third-generation designs (BE3)
- Cytidine deaminase converts C>U within accessible ssDNA on the non-target strand
- D10A nickase Cas9 cleaves the non-edited DNA strand which directs cellular DNA repair to replace the G-containing DNA strand
- The deaminated strand is used to template the repair to produce a U:A base pair (long-patch base excision repair)
- This intermediate then converted to T:A during DNA replication
How can you improve the efficiency of C>T base editing? (4)
- Fuse a uracil glycosylase inhibitor (UGI) to nickase which blocks uridine excision and subsequent base excision repair
- Nickase dCas9 D10A nicks the target strand opposite the deaminated cytidine
- Initiates long-patch base excision repair where the deaminated strand is used to template the repair producing a U:A base pair
- Intermediate is then converted to T:A during DNA replication
What is the function of cytidine deaminase?
Converts C>U
What is prime editing? (4)
- Alternative method of base editing
- Nickase Cas9 (H840A) is fused to engineered reverse transcriptase (RT) enzyme
- sgRNA is extended to encode a guide RNA and a repair template called a prime editing guide RNA (PEG RNA) containing the mutation
- RT creates cDNA based on RNA template sequence and is incorporated into nicked target site
