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BIS477 World of RNA > Editing the genome: Mechanisms of CRISPR-Cas9 > Flashcards

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)
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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
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3
Q

How are Cas9-induced DSBs repaired? (2)

A
  • Non-homologous end-joining (NHEJ) or
  • Homology-directed repair
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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
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5
Q

What is HDR? (2)

A
  • Homology-directed repair
  • Donor template is used for gene correction of knock-in experiments
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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
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7
Q

What is TIDE? (2)

A
  • Tracking of indels by decomposition (computational analysis)
  • Sanger trace of edited will be disrupted compared to WT
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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

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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

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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
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11
Q

How can you sort for successfully transfected cells?

A

Use GFP-tagged Cas9

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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
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13
Q

What is the PAM site? (2)

A
  • Protospacer adjacent motif
  • Determines where the cleavage occurs in the DNA
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14
Q

What are the nuclease domains of Cas9? (3)

A
  • HNH (target strand)
  • RuvC (non-target strand)
  • Function independently of each other
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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
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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
Q

What are methods of using dCas9 for gene regulation? (5)

A
  • CRISPRa
  • CRISPRi
  • SunTag system
  • Epigenome editing
  • Base editing
26
Q

What is CRISPRa? (3)

A
  • CRISPR activation
  • Fusion of dCas9 with potent transcription factor VP64
  • Transcriptional activation of a specific gene without editing
27
Q

What is CRISPRi? (3)

A
  • 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
Q

What is the SunTag system? (2)

A
  • 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
Q

What is epigenome editing with dCas9? (3)

A
  • 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
Q

What is base editing? (3)

A
  • 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
Q

How does base editing work? (3)

A
  • 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
Q

How does C>T base editing work? (5)

A
  • 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
Q

How can you improve the efficiency of C>T base editing? (4)

A
  • 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
Q

What is the function of cytidine deaminase?

A

Converts C>U

35
Q

What is prime editing? (4)

A
  • 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

BIS477 World of RNA (14 decks)

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