Genome editing

Question 1

What 2 things does genome editing combine?

Answer 1

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

Question 2

What are the 4 types of engineered nucleases?

Answer 2

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

Question 3

What are meganucleases?

Answer 3

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

Question 4

What are ZFNs?

Answer 4

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

Question 5

What are TALENS?

Answer 5

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

Question 6

What is CRISPR/Cas9?

Answer 6

Clustered Regularly Interspaced Short Palindromic Repeat/ CRISPR associated protein 9

Question 7

What are the two classes of CRISPR/Cas9?

Answer 7

1. Class I uses multiple Cas proteins

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

Question 8

Where is Cas9 derived from?

Answer 8

Streptococcus pyogenes

Question 9

What is the Cas protein and what does it do?

Answer 9

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

Question 10

What does Cas9 require in order to induce DNA cleavage?

Answer 10

A protospacer adjacent motif (PAM)

Question 11

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

Answer 11

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

Question 12

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

Answer 12

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

Question 13

What does HDR CRISPR/Cas9 entail?

Answer 13

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

Question 14

What does a dual nickase strategy do?

Answer 14

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

Question 15

What can point mutations with CRISPR/Cas9 do?

Answer 15

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

Question 16

What are some modifications of CRISPR?

Answer 16

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

Question 17

What are some current Monogenic diseases benefiting from gene editing?

Answer 17

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

Question 18

What mutation causes Duchenne Muscular Dystrophy?

Answer 18

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

Question 19

What is DMD?

Answer 19

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

Question 20

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

Answer 20

• 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

Question 21

What are some ethical considerations of CRISPR?

Answer 21

• eugenics
• misapplication
• inequitable access
• regulation
• embryo modification

Question 22

What are the steps in designing a CRISPR experiment?

Answer 22

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

Question 23

What is the process in selecting CRISPR approach?

Answer 23

• 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

Question 24

What is the process of designing a gRNA?

Answer 24

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

Question 25

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

Answer 25

- Expression plasmid - Viral vectors (Lenti virus and AAV) - Ribonucleoprotein (RNP) complex

Question 26

What are the pros and cons of plasmid delivery?

Answer 26

Pros: easy to use, cheap, easy to customise, stable, non-toxic Cons: Transient, some cell lines difficult to transfect, mostly only in vitro

Question 27

What are the pros and cons of RNP delivery?

Answer 27

Pros: quick, less off-targets Cons: larger proteins hard to make, more expensive than plasmid

Question 28

What are the pros and cons of Lenti virus delivery?

Answer 28

Pros: more efficient for hard to transfect cells, stable integration, lots of flexibility Cons: Integrates into DNA, safety, more training and approvals needed

Question 29

What are the pros and cons of AAV delivery?

Answer 29

Pros: safe, selective tropism, non-integrating Cons: very small packaging limit

Question 30

What needs to be considered when picking delivery system?

Answer 30

- Cell type - size of constructs - longevity of Cas9 expression - selection?

Question 31

What is the process of sorting and selecting cells?

Answer 31

- Sort by fluorescence signals? - single cell sort by dilution - select positively transfected cells with antibiotic selection

Question 32

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

Answer 32

- 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

Question 33

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

Answer 33

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

Question 34

What is the process for checking for off-targets?

Answer 34

- 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

Question 35

What are the methods used to predict off targets?

Answer 35

- Heteroduplex analysis - Circle-seq - Guide-seq - BLISS

Question 36

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

Answer 36

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)

Question 37

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

Answer 37

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?)

Question 38

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

Answer 38

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

Question 39

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

Answer 39

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

Question 40

Can you check for off-targets with dCas9?

Answer 40

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

Question 41

Controls of CRISPR

Answer 41

- cells need to be treated exactly the same | - does the process of single cell cloning/editing alter how the cells behave (karyotype)?