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GENE3340 MOLECULAR GENETICS > Genome editing > Flashcards

Genome editing Flashcards

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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
Q

What are the different delivery systems for CRISPR/Cas 9 and gRNA?

A
  • Expression plasmid
  • Viral vectors (Lenti virus and AAV)
  • Ribonucleoprotein (RNP) complex
26
Q

What are the pros and cons of plasmid delivery?

A

Pros: easy to use, cheap, easy to customise, stable, non-toxic

Cons: Transient, some cell lines difficult to transfect, mostly only in vitro

27
Q

What are the pros and cons of RNP delivery?

A

Pros: quick, less off-targets

Cons: larger proteins hard to make, more expensive than plasmid

28
Q

What are the pros and cons of Lenti virus delivery?

A

Pros: more efficient for hard to transfect cells, stable integration, lots of flexibility

Cons: Integrates into DNA, safety, more training and approvals needed

29
Q

What are the pros and cons of AAV delivery?

A

Pros: safe, selective tropism, non-integrating

Cons: very small packaging limit

30
Q

What needs to be considered when picking delivery system?

A
  • Cell type
  • size of constructs
  • longevity of Cas9 expression
  • selection?
31
Q

What is the process of sorting and selecting cells?

A
  • Sort by fluorescence signals?
  • single cell sort by dilution
  • select positively transfected cells with antibiotic selection
32
Q

What is the process of screening and validating in HDR base editor approach?

A
  • 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
Q

What is the process of screening via Gene KO (creating indels)?

A

PCR around your region of interest, perform Sanger of your amplicon, analyse proportion of amplicons that have been edited using TIDE software

34
Q

What is the process for checking for off-targets?

A
  • 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
Q

What are the methods used to predict off targets?

A
  • Heteroduplex analysis
  • Circle-seq
  • Guide-seq
  • BLISS
36
Q

What is Heteroduplex analysis? and what are the pros and cons?

A

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
Q

What is circle-seq analysis? and what are the pros and cons?

A

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
Q

What is guide-seq analysis? and what are the pros and cons?

A

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
Q

What is BLISS? and what are the pros and cons?

A

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
Q

Can you check for off-targets with dCas9?

A
  • YES
  • done with chromatin immunoprecipitation (ChIP)
  • genomic sites which are bound by Cas9 are pulled down with a Cas9 antibody and sequenced
41
Q

Controls of CRISPR

A
  • 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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