RNA EDITING

Question 1

What is RNA editing

Answer 1

enables scientists to change the molecules that carry the instructions needed to produce proteins without changing the original DNA code.

MAY INCLUDE - insertion, deletion and substitution of nucleotides. (any thing other then splicing that changes RNA transcript)

Question 1

RNA editing types (3)

Answer 1

1. Adenosine deaminase actine on RNA (ADAR)
2. Cas13
   CRISPR Cas-inspired RNA targeting system (CIRTS)

Question 2

What is ADAR (4)

Answer 2

RNA-binding protein.

• functions in RNA editing through post-transcriptional modification of mRNA transcripts by changing the nucleotide content of RNA.
• ADARs are expressed in human cells.
• ADAR family: ADAR1, ADAR2 (aka ADARB1) and ADAR3 – only 1 and 2 have catalytic activity.

Question 3

ADAR CONVERSIONS

Answer 3

A to I in the RNA disrupts A:U pairing - making RNA unstable.

Inosine is structurally similar to guanine - which leads to I-to-C binding.

A to I deamination is the most abundant form of RNA editing in mammals.

Question 4

Treatment ADAR for muscular dystrophies

Answer 4

AAV8 mediated ADAR RNA editing for MDx mouse model.
- nonsense mutation

• target Mdx mice via CRISPR-Cas9 excision of exon 23.
• ADAR1 double and used synthetic ADAR1 they could increase editing efficiency more then any other groups.

Question 5

Cas13

Answer 5

• like Cas9, its a novel type of RNA-targeting enzyme
• The cas13 protein complexes with the guide RNA via recognition of a short hairpin in the crRNA – and target specific that is complementary to a target region.

Question 6

Cas13 orthologs?

Answer 6

Cas13d – which has been leveraged for efficient and robust knockdown across many endogenous transcripts.

• Cas13d – used to modulate splicing of endogenous transcripts and that coding sequence for Cas13d is small enough to fit within packaging limits of AAV delivery.
• Cas13a / b/ c – too big. Which decreases deficiency due to dual delivery.

Question 7

what has dCas13 been used for:

Answer 7

Has been used to target FTD with Parkinsonism linked to chromosome 17 – autosomal dominant major neurodegenerative disease caused by diverse point mutation in MAPT (encodes for tau).
- via AAV

Question 8

what is REPAIR?

Answer 8

RNA editing for programmable A to I replacement (REPAIR) – works by fusing ADAR2 deaminase domain to Cas13b.

Question 9

Disadvantages of REPAIR (3)

Answer 9

• Our body has naturally occurring Abs to CRISPR-Cas proteins.
• Also large size is challenging for packaging into a translation viral vector for direct protein delivery (130kDa in size)
• OVERCOME with CIRTS

Question 10

what is CIRTS

Answer 10

CRISPR-Cas inspired RNA targeting system (CIRTS)
- Naturally occurring CRISPR-Cas programmable RNA binding system - made from human proteins
- avoids immune issues.

Question 11

CIRT-6

Answer 11

Using CIRT-6 (optimal confirmation effector protein) – they could package entire thing into an AAV – making it much more appealing.

This is a good example of where you are removing bacterial components (using humanised protein) – no immune response, reducing RNA (by 50%), AND you can put it into an AAV.