Editing the genome: Mechanisms of CRISPR-Cas9 Flashcards
1
Q
What are the components of the CRISPR-Cas9 system? (2)
A
- Guide RNA (gRNA)
- CRISPR associated endonuclease (Cas9)
2
Q
What is gRNA? (3)
A
- 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
3
Q
How are Cas9-induced DSBs repaired? (2)
A
- Non-homologous end-joining (NHEJ) or
- Homology-directed repair
4
Q
What is NHEJ? (2)
A
- Non-homologous end joining
- Results in insertions/deletions (indels) that are often exploited to create frameshift or knockout mutations
5
Q
What is HDR? (2)
A
- Homology-directed repair
- Donor template is used for gene correction of knock-in experiments
6
Q
How do you validate genome edits? (7)
A
- 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
7
Q
What is TIDE? (2)
A
- Tracking of indels by decomposition (computational analysis)
- Sanger trace of edited will be disrupted compared to WT
8
Q
How can you easily identify cells which have integrated the donor sequence?
A
Encode a restriction site in the repair template so the PCR product can be cleaved to prove integration e.g. NheI
9
Q
What is the efficiency of HDR?
A
HDR is very low efficiency (<10% of modified alleles) as NHEJ pathways tend to be favoured over HDR
10
Q
How can HDR efficiency be improved? (6)
A
- 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
11
Q
How can you sort for successfully transfected cells?
A
Use GFP-tagged Cas9
12
Q
How does DNA cleavage occur? (4)
A
- 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
13
Q
What is the PAM site? (2)
A
- Protospacer adjacent motif
- Determines where the cleavage occurs in the DNA
14
Q
What are the nuclease domains of Cas9? (3)
A
- HNH (target strand)
- RuvC (non-target strand)
- Function independently of each other
15
Q
How does the HNH domain work? (2)
A
- Uses a one metal ion mechanism to hydrolyse the scissile phosphates in the target strand backbone
- Active site has 3 catalytic residues
16
Q
What are the 3 catalytic residues of the catalytic pocket of HNH?
A
- Asp839
- His840
- Asn863
17
Q
How does the RuvC domain work? (2)
A
- Uses a two-metal ion mechanism to hydrolyse the scissile phosphates in the non-target strand backbone
- Active site has 4 catalytic residues
18
Q
What are the 4 catalytic residues of the catalytic pocket of RuvC?
A
- Asp10
- Glu76
- His983
- Asp986
19
Q
What is dCas9?
A
Catalytically dead Cas9
20
Q
What are the features of dCas9? (3)
A
- 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
21
Q
Why is dCas9 useful?
A
Change the catalytic activity but can still target it to specific DNA sequences because of the gRNA
22
Q
What is Cas9 nickase?
A
Single mutant of HNH or RuvC domain so only causes a single strand break
23
Q
What is the main problem with CRISPR-Cas9?
A
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
24
Q
How can you limit CRISPR-Cas9 off-target effects? (3)
A
- ‘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
25
What are methods of using dCas9 for gene regulation? (5)
- CRISPRa
- CRISPRi
- SunTag system
- Epigenome editing
- Base editing
26
What is CRISPRa? (3)
- CRISPR activation
- Fusion of dCas9 with potent transcription factor VP64
- Transcriptional activation of a specific gene without editing
27
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
28
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
29
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
30
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
31
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
32
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
33
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
34
What is the function of cytidine deaminase?
Converts C>U
35
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
