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GENE3340 MOLECULAR GENETICS > Genome editing > Flashcards

Genome editing Flashcards

1
Q

What 2 things does genome editing combine?

A

A DNA cleavage domain (non-specific) with a DNA binding molecule (sequence specific) –> engineered nuclease

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2
Q

What are the 4 types of engineered nucleases?

A
  1. Engineered meganucleases
  2. ZFN
  3. TALENS
  4. CRISPR/Cas9
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3
Q

What are meganucleases?

A

Engineered versions of naturally occuring REs. Have extended DNA recognition sequences and contain few sites within the human genome. Difficult to engineer to create specific DNA binding for a new target site

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4
Q

What are ZFNs?

A
  • Zinc Finger Nucleases
  • most common DNA binding motif in euks
  • ZF domain consists of 30 amino acids
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5
Q

What are TALENS?

A

Transcription activation-like effector nucleases. Naturally occurring protein from plant pathogenic bacteria. Contains DNA binding domains that each recognise a single base pair

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6
Q

What is CRISPR/Cas9?

A

Clustered Regularly Interspaced Short Palindromic Repeat/ CRISPR associated protein 9

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7
Q

What are the two classes of CRISPR/Cas9?

A
  1. Class I uses multiple Cas proteins

2. Class II uses a single large Cas (e.g. Cas9)

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8
Q

Where is Cas9 derived from?

A

Streptococcus pyogenes

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9
Q

What is the Cas protein and what does it do?

A

It is a DNA endonuclease which cleaves dsDNA when guided to a specific DNA target by a bound guide RNA

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10
Q

What does Cas9 require in order to induce DNA cleavage?

A

A protospacer adjacent motif (PAM)

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11
Q

What are the 2 endogenous repair mechanisms induced by DNA cleavage?

A
  1. NHEJ (Nonhomologous end joining): error-prone, most common
  2. HDR (Homology directed repair): not error prone (results in specific repair of DNA), only found in dividing cells
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12
Q

What does the traditional cut and paste CRISPR/Cas9 entail?

A

Cutting DNA at target loci can be used to induce mutations by NHEJ. Very useful for making KO cell lines and animal models. Easy to do and efficient

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13
Q

What does HDR CRISPR/Cas9 entail?

A

Introduction of a repair template can be used to insert new DNA sequences by HDR. Very low efficiency. Highly desirable from a therapeutic perspective.

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14
Q

What does a dual nickase strategy do?

A

Less off targets and increases prevalence of HDR (more specific approach)

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15
Q

What can point mutations with CRISPR/Cas9 do?

A
  • create a high fidelity (Cas9-HF1) and enhanced binding specificity (eCas9)
  • fewer off targets/no off targets
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16
Q

What are some modifications of CRISPR?

A
  • tagged CRISPR for live imaging?
  • gene regulation (gene activation/repression domains added)
  • base editing (deaminates of nts)
  • inducible systems? - responding to only certain stimuli
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17
Q

What are some current Monogenic diseases benefiting from gene editing?

A
  • Cystic fibrosis
  • haemophilia
  • sickle cell disease
  • duchenne muscular dystrophy (DMD)
18
Q

What mutation causes Duchenne Muscular Dystrophy?

A

Caused by mutations in the gene for dystrophin on the X chromosome. Mutations that delete exon 44 of dystrophin most common cause

19
Q

What is DMD?

A

Dystrophin is a large cytoskeletal structural protein essential for muscle cell membrane integrity (affects 1 in 3500 boys)

20
Q

What are the limitations of CRISPR for human therapy and potential solutions?

A
  • Packing limit
    • use split Cas9? Or alternative Cas proteins
  • off-target effects
    • high fidelity Cas9’s have less off-target/none
  • immune reactions to Cas9
    • edit cells outside body then return edited cells which no longer express Cas9
21
Q

What are some ethical considerations of CRISPR?

A
  • eugenics
  • misapplication
  • inequitable access
  • regulation
  • embryo modification
22
Q

What are the steps in designing a CRISPR experiment?

A
  1. select CRISPR approach
  2. Design gRNA to other components and test that they cut DNA
  3. deliver gRNA and Cas9 to target
  4. screen, sort, select cells
  5. validate edit/effect
  6. check for off-target effects
23
Q

What is the process in selecting CRISPR approach?

A
  • What do you want to achieve? Basic versus human therapy? Correction? Upregulation? Deletion - allele specific or conditional? Tracking of cellular location?
  • does it need to be derived from human cells (delivery type)? Is the cell dividing or not? How large is the machinery you want to deliver?
  • is efficiency or specificity more important?
  • Is DNA or RNA the target
24
Q

What is the process of designing a gRNA?

A

Ensure that your gene does not overlap with other genes. Use software to predict off target effects. Design more than one set - very locus specific, also different gRNA with same target can act as controls

25
What are the different delivery systems for CRISPR/Cas 9 and gRNA?
- Expression plasmid - Viral vectors (Lenti virus and AAV) - Ribonucleoprotein (RNP) complex
26
What are the pros and cons of plasmid delivery?
Pros: easy to use, cheap, easy to customise, stable, non-toxic Cons: Transient, some cell lines difficult to transfect, mostly only in vitro
27
What are the pros and cons of RNP delivery?
Pros: quick, less off-targets Cons: larger proteins hard to make, more expensive than plasmid
28
What are the pros and cons of Lenti virus delivery?
Pros: more efficient for hard to transfect cells, stable integration, lots of flexibility Cons: Integrates into DNA, safety, more training and approvals needed
29
What are the pros and cons of AAV delivery?
Pros: safe, selective tropism, non-integrating Cons: very small packaging limit
30
What needs to be considered when picking delivery system?
- Cell type - size of constructs - longevity of Cas9 expression - selection?
31
What is the process of sorting and selecting cells?
- Sort by fluorescence signals? - single cell sort by dilution - select positively transfected cells with antibiotic selection
32
What is the process of screening and validating in HDR base editor approach?
- can screen via Restriction digest - can screen by NGS - more expensive; slower - good for when screening lots of different single cell clones at once, each clone tagged with barcode - gives precise info
33
What is the process of screening via Gene KO (creating indels)?
PCR around your region of interest, perform Sanger of your amplicon, analyse proportion of amplicons that have been edited using TIDE software
34
What is the process for checking for off-targets?
- off target prediction software - assign an on-target and an off-target score which predicts how good your gRNA will be - produces list of candidate locations which will likely be cut by your gRNA
35
What are the methods used to predict off targets?
- Heteroduplex analysis - Circle-seq - Guide-seq - BLISS
36
What is Heteroduplex analysis? and what are the pros and cons?
Must perform PCR on region of interest, then denature PCR products and anneal them back together. If no mutations makes homoduplex. If mutations makes heteroduplexes. Can run on a gel, use melt analysis or PAGE to detect changes. Pros: quick, cheap, requires no specialised expertise Cons: requires pre-conceived prediction of off-targets (biased)
37
What is circle-seq analysis? and what are the pros and cons?
Assay performed in a test tube. Extract DNA from cell of interest, mix with Cas9 and guide. Where gDNA cut, now have linear fragments (can circularise those) and can be analysed by NGS. Pros: unbiased, sensitive, requires only 4-5 million reads Cons: in vitro (still need in vivo?)
38
What is guide-seq analysis? and what are the pros and cons?
Guide seq is where ds oligodeoxynucleotide tags are inserted at dsDNA breaks (in live cells). Tags where Cas9 cuts DNA, adapters added. Pros: unbiased, performed in living cells, captures breaks over time Cons: sequencing depth defines sensitivity - cost, requires high cell number
39
What is BLISS? and what are the pros and cons?
Cells/tissues fixed. Cut the dna, and in promoter, transcribe the promoter (allows amplification to find where cut sites are). Uses barcodes and T7 Pros: unbiased, in situ, can be performed on tissues, applicable for low cell number Cons: Susceptible to fixation artifacts, captures only a single time point
40
Can you check for off-targets with dCas9?
- YES - done with chromatin immunoprecipitation (ChIP) - genomic sites which are bound by Cas9 are pulled down with a Cas9 antibody and sequenced
41
Controls of CRISPR
- cells need to be treated exactly the same | - does the process of single cell cloning/editing alter how the cells behave (karyotype)?

GENE3340 MOLECULAR GENETICS (20 decks)

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