Targeted Gene Modification - 2

Front 1

What is the issue with using most transposons for targeted gene modification?

Back 1

Using regular transposons for gene therapy presents the issue that when transposons are excised and move they leave remnants of small unwanted excision sequences in the DNA which can interfere with surrounding gene regulatory elements

Front 2

What transposon can be used for targeted gene modification?

What advantage does it have over regular transposons?

Back 2

A moth transposon 'PiggyBac' does not leave remnant sequences in the genome which becomes cleanly removed from the genome which it has transposed onto.

This transposon can be modified in order to correct unwanted mutations in stem cells.

Front 3

Where are TALENs sourced from?

Back 3

Plant pathogenic bacteria which act as effectors during pathogenesis.

Front 4

Describe the structure and function of TALEN subunits.

Back 4

Contain an N terminus translocation signal and a transcriptional activation domain. The Tal effector has an area which contains multiple 33-35 DNA binding repeats - These give the TALE its specificity.

Front 5

What confers the specificity of a TALEN?

Back 5

33-35 AA DNA binding repeats which bind to a single base pair each.

This is decided by 2 hyper variable AAs at positions 12-13 in each repeat sequence which recognise specific bases in target promoters.

Front 6

What are the 3 steps of action of the CRISPR - cas system?

Back 6

1. Acquisition

2. Expression

3. Interference

Front 7

Briefly describe the acquisition step of the CRISPR - cas9 system.

Back 7

Integration of foreign DNA as a protospacer at the leader site of the CRISPR locus

Front 8

Briefly describe the expression stage of the CRISPR - cas9 system.

Back 8

Foreign DNA expressed and made into crispr RNAs (crRNAs) by cas proteins and trans activating crRNA`

Front 9

Briefly describe the interference stage of the CRISPR-cas9 system.

Back 9

crRNA and trans encoded crRNA specifically direct the cas-9 nuclease to cleave foreign DNA.

Front 10

How is the CRISPR - cas9 system altered artificially?

Back 10

The 2 cas 9 guidance RNAs are fused together to form sgRNA - which is able to form an RNA guided endonuclease that mediates sequence specific cleavage in the genome.

Front 11

Give an example of how the CRISPR - cas system can be used in a human disease scenario.

Back 11

The CCR5 gene in humans encodes critical coreceptors for the pathogenesis of HIV.

This can be inactivated in human hematopoietic stem and effector cells (HSPCs) using the CRISPR / cas system to recognise a 20 bp sequence.