New and Future Developments

Question 1

What are the current methods for both reading and writing the genome and how are they beneficial?

Answer 1

Reading: Fast and affordable DNA/RNA sequencing methods which lead to continued expansion of different “omics” databases
Writing: Efficient, precise, versatile genetic modification methods

Question 2

What are the benefits of Next Generation Sequencing?

Answer 2

Allows the sequencing of a genome in a few hours for under £1000. 3rd generation methods focus on sequencing the genome from small amounts of DNA using 10-100kb sequencing reads. Long read are more likely to incorporate the whole of a repetitive region - which means the DNA sequence is more accurately assembled with less gaps

Question 3

Describe the benefits of Single Cell Sequencing

Answer 3

Sequencing usually requires large amounts of DNA (multiple cells). A new method allows individual cells to be sequenced. Single cell RNA-seq is revealing cellular heirarchies and previously unknown cell types in tissues. We can use this to analyse tumour biopses over time to reveal much about the tumour evolution before and after therapy

Question 4

What are the limits of gene editing and CRISPR-based methods

Answer 4

Specificity - Cas9 may cut off target
Safety - Can result in genome damage (translocations)
Efficiency - HR edits usually swamped by NHEJ
Versatility - Editing restricted to target loci with an adjacent PAM

Question 5

How can we address the specificity limitation of CRISPR methods?

Answer 5

Use online tools to search for genomic sequences that match gRNA and use the ones with the least off target effects. Also, the GC content of the DNA:gRNA heteroduplex can affect off target cleavage. Position of gRNA:DNA mismatches relative to PAM. Chromatin status of gene target. Sequence of scaffold RNA.

Question 6

How can we address the safety limitation of CRISPR methods?

Answer 6

DSBs can lead to large insertions or deletions and translocations. This depends on the alternative NHEJ pathway. We need to identify and temporarily suppress the alternative NHEJ pathway. Or, we can avoid DSBs completely - by fusing inactive Cas9 to a base editor like cytosine deaminase. This converts CG bp at target site to AT, editing specific bases as needed

Question 7

How can we address the efficiency limitation of CRISPR methods?

Answer 7

We can encourage homology directed repair by inhibiting NHEJ

Question 8

How can we address the versatility limitation of CRISPR methods?

Answer 8

CRISPR Cas systems from other bacteria and archae can lead to more choice of CRISPR targets due to their different PAM sequences

Question 9

Describe the idea and uses of Gene Therapy

Answer 9

Gene therapy is introducing a therapeutic gene into a patient so that the gene is stably maintained. This could result in a lifetime cure to a disease from a one-off treatment. It can be used to treat monogenic, viral diseases, and cancer. It is not always necessary for the gene to integrate into the genome (if the cells are non-dividing) but the gene will always need to go into the nucleus to be expressed. For target tissues with a high turnover, the gene will need to integrate into the stem cell genome - CRISPRing HSC can cure X-linked severe combined immunodeficiency

Question 10

How is gene therapy performed in stem cell populations?

Answer 10

The patients stem cells are removed and cultured, and the treatment gene is added to a harmless (normal genes removed) virus and is introduced to the stem cells which are then returned to the patient

Question 11

How can gene therapy result in cancer?

Answer 11

Integration of the retroviral genome close to a proto-onco gene. Retroviral genomes contain long terminal repeat sequences with strong enhancers that can upregulate host genes close to their integration site.

Question 12

What are the advantages of gene therapy?

Answer 12

Safety - Less chance of random gene integration leading to oncogenic activation
Efficiency - Corrected gene more likely to restore normal expression than a randomly integrated extra gene
Versatility - Genes edited in specific ways such as inactivating dominant genes

Question 13

What is pharmacogenomics? Why is it necessary?

Answer 13

“Before anyone is prescribed a drug, their genetic info will be looked at to determine the right drug, at the right dose, at the right time”. The NHS currently spends so much on drugs, and 90% of drugs only work in 30-50% of the population. 6.5% of hospital admissions are from adverse drug reaction

Question 14

How can personalised medicine help in treating in tumours?

Answer 14

Almost every tumour is genetically unique. Lung cancer tumours positive for EGFR-TK gene are more likely to respond to certain drugs. Therefore, we need to determine which mutations lead to sensitivities to which drugs.

Question 15

Why does personalised medicine not always lead to success?

Answer 15

Cancers becoming resistant is a huge problem. In melanoma, tumours with activating mutations in BRAF gene respond well to B-raf inhibitors, but they become resistant - we need to research why

Question 16

Explain the concept of risk, screening and prevention

Answer 16

Risk - the probability that a patient gets a disease within a given time
Screening - testing individuals to determine their risk levels or for diagnosis
Prevention - reducing risk for high risk patients by drugs, lifestyle changes, etc.

Question 17

How can we gain more insight into risk?

Answer 17

Studying large numbers of genomes allows better understanding of the intrinsic genetic components of risk. We need to evaluate the combinatorial effects of different factors to have a comprehensive understanding

Question 18

How can we use genome engineering to make better antibody drugs?

Answer 18

We raise the antibodies in animals, but they must be humanised so that they work effectively. We do this by adding human antigen binding components onto the non human framework. This is hard to do without impairing the binding affinity. However, we can engineer mice so that their array of antibody genes has been partly or wholly humanised - mouse makes high affinity humanised antibodies

Question 19

Describe how genetic engineering in pigs is beneficial for humans

Answer 19

Pigs have genes whose products can promote rejection after transplantation. Also, the pig genome contains porcine endogenous retrovirus which may be reactivated and infect human cells. We can engineer the pig cells to be compatible with the human immune system and use their organs for xenotransplantation.

Question 20

How can we eradicate malaria using gene drives?

Answer 20

Gene drive is where an allele is preferentially transmitted during meiosis so that it spreads throughout the population. If we make one allele in a mosquito cause infertility and codes for Cas9 which cuts the WT allele in meiosis and repairs using HR with infertility gene as a template, creating homozygous germ cells. This spreads the gene rapidly.

Question 21

List some of the aspects of ageing

Answer 21

Altered intercellular communication
Genomic instability
Telomerase attrition
Epigenetic alterations
Loss of proteostasis
Deregulated nutrient sensing
Mitochondrial dysfunction
Cellular senescence
Stem cell exhaustion

Question 22

How do mutations impact ageing?

Answer 22

It is thought that accumulating genome damage in somatic cells underlies the declining physiology of ageing. This is consistent with the fact that mutations do accumulate in ageing somatic cells. Patients with rare early ageing syndromes have mutations in their DNA repair protein genes

Question 23

How do telomeres relate to ageing?

Answer 23

As we age, our telomeres shorten which puts our genome at risk of instability. We could upregulate telomerase, replenishing telomeres. However, this can greatly increase the risk of certain cancers

Question 24

List some of the ethical issues behind genetic engineering

Answer 24

Designer babies

Germline effects, ie consent, do we know all the risks, etc