Genetic therapies for neurological disease

Question 1

What is gene editing

Answer 1

the process of converting a targeted DNA sequence into a new desired DNA sequence in the native context of the cell’s genome.

Question 2

CRISPR and microbial immunity:

Answer 2

• CRISPR was discovered as part of the adaptive immune system in bacteria and archaea.
• CRISPR/Cas systems destroy invading nucleic acids (viruses or plasmid) via endonuclease activity.
• The type II CRISPR system (with Cas9) is mostly used in research and therapeutics. As it only involves on Cas9 nuclease – the others require multiple complexing.

Question 3

What is PAM

Answer 3

PAM = PROTOSPACER ADJACENT MOTIF
- 3 nucleotide consensus sequence immediately 3’ of the protospacer. required for the CRISPR/Cas9 to cut the DNA
- for spCas9 this is NGG

Question 4

Minimal three components originally needed for gene editing

Answer 4

1. tracrRNA
2. crRNA
3. Cas9 endonuclease

Question 5

Cas9 endonuclease mechanism

Answer 5

1. Cas9 binds PAM
2. Cas9 unwinds DNA duplex 3’-5’ direction of PAM
3. sgRNA spacer anneals to photospacer
4. The displaced strand forms a single-stranded DNA loop
5. conformational changes activates endonuclease domains. RuvC cuts PAM strand.
   both DNA strands are cut 3 nts upstream of the PAM within the photospacer - generating a blunt double-strand break.

Question 6

Original Cas9 protein:

Answer 6

• has two endonuclease domains that are active and cause a double standard DNA break

Question 7

Cas9 mutated?

Answer 7

1. mutating one of the endonucelases - you create Cas9 nickase (Cas9n) - which only cuts ONE DNA strand.
2. mutating both the endonucleases - you create dead Cas9 (dCas9) - does NOT cut DNA.

Question 8

How are dCas9 and Cas9n formed?

Answer 8

fusing Cas9 protein to different protein domains.

Question 9

How does cas9n work?

Answer 9

nickase.
new effector funciton:
- base editors fused to nucleotide deaminase.
- prime editors fused to reverse transcriptase.

Question 10

How does dCas9 work?

Answer 10

Modifies regulation of gene expression.
- CRISPR activation: increased expression, fused to transcription activator
- CRISPR inhibition: decreases expression, fused to transcription repressor
- Epigenome editors: can also fuse to various types to also regulate gene expression by aletring the confirmation of chromatin to make it less accessible to TFs.

Question 11

Rational design VS. directed evolution?

Answer 11

rational design: computer aided and site-directed mutagenesis - to determine engineered Cas

Directed evolution: random mutagenesis in cells to achieve evolved Cas.

Question 12

Base editors

Answer 12

These are nucleotide deaminase domains fused to a Cas9n.
- these enable the engineering of a single base pair change WITHOUT DBS.
- Nickase stimulates the DNA repair pathway - in a way that increases editing efficiency.

Question 13

THE MAIN BASE EDITORS

Answer 13

1. CBE : converts C to T (via uracil) using a cytidine deaminase domain
   ABE : converts A to G (via inositol) using an adenosine deaminase domain.

Question 14

PRIME EDITORS

Answer 14

very versatile.

• PEs are reverse transcriptases fused to Cas9n
• engineered single base pair changes, small insertions and deletions.
• uses a specific prime editing gRNA (PEGRNA) containing desired new sequences to be installed.

Question 15

CRISPR examples of transcriptional activators: (4)

Answer 15

1. VP64
2. VPR
3. SunTag
4. SAM

Question 16

CRISPRi - transcriptional repressor most commonly used

Answer 16

Krüppel-associated box (KRAB)

Question 17

how was CRISPR discovered?

Answer 17

bacteria/archaea

Question 18

the classical CRISPR/Cas9 system uses …

Answer 18

Cas9 endonucleases from Streptococcus pyogenes (spCas9).

Question 19

TWO main outcomes of CRISPR/Cas9 gene editing:

Answer 19

1. non-homologous end-joining
2. homology-directed repair

Question 20

what is the NHEJ?

Answer 20

In the NHEJ pathway, random insertions and deletions occur at the cleavage site, leading to error-prone repair.
It is effectively the cell panicking – it is quickly ‘sticking’ DNA back together making it error prone – to indels.

Question 21

WHAT IS HDR?

Answer 21

Homology directed repair.

HDR is a precise mechanism where homologous chromosomal DNA acts as a guide for damaged DNA during repair, resulting in minimal editing errors.

Question 22

Advantages/disadvantages of HDR

Answer 22

+ precise

• inefficient
• cannot target post-mitotic cells (non-dividing)

Question 23

Advantages/disadvantages of NHEJ

Answer 23

+ efficient
+ post-mitotic cells i.e. neurons

• error prone - indels

Question 24

What are indels?

Answer 24

random nucleotide insertions or deletions at the cut site. Causing disrupted DNA. As a result CRISPR is very good at producing precise gene knock outs – BUT from a therapeutic perspective this would not be ideal.

Question 25

Homology-independent targeting integration (HITI)

Answer 25

Involves exogenously providing a double-stranded DNA template.

Question 26

Prime / base editors - DSB?

Answer 26

PEs and BEs do not require DSBs.

Question 27

what is the MMR pathway?

Answer 27

mismatch repair pathway - influences editing outcomes in both BE and PE in different ways.

• IN PE - MMR reduces efficiency
• IN BE - MMR can insert the correct edit.

Question 28

Loss-of-function

Answer 28

mutant gene lacks function of the WT.

Often caused by haploinsufficiency
CRISPRa appraoch

Question 29

Gain-of-function

Answer 29

mutant gene has enhanced function or expression of WT.

CRISPRi approach.

Question 30

Challenges facing CNS gene editing: (3)

Answer 30

1. delivery
2. efficiency
3. safety

Question 31

Split intein Cas9

Answer 31

Split Cas9 gene editor into two and fuse it to intein.

INTEIN = protein splicing domain

if the cell is co-transduced, you will get an expression of the end terminal and c terminal portion of the editor and intein domains. INCREASES EFFICIENCY.

Question 32

What are VLPs?

Answer 32

VIRUS LIKE PARTICLES.

• assemblies of viral proteins that can infect cells but lack viral genetic material
• they have an advantage of transient expression with the advantages of viral targeting
• failed to target many cells.

Question 33

What is mosaicism?

Answer 33

Mosaicism occurs when a person has two or more genetically different sets of cells in his or her body. If those abnormal cells begin to outnumber the normal cells, it can lead to disease that can be traced from the cellular level to affected tissue, like skin, the brain, or other organs.