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Genetics > GENE 10: New and future developments > Flashcards

GENE 10: New and future developments Flashcards

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1
Q

What is the main benefit of NGS?

A

Effectively reduces the costs and time required to sequence the human genome

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2
Q

What are third generation sequencing methods intending to do?

A

seeking to improve NGS methods by providing longer sequence reads – 10-100kb versus 250 kb – using smaller amounts of DNA.

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3
Q

Why are long-reads for sequencing desirable?

A

They are more likely to incorporate whole tandemly repeated regions with fewer gaps in the genome sequence. They also facilitate haplotype phasing which is the determination of whether sequence variations or methylations are on the same or different chromosomes.

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4
Q

What is SMRT sequencing based on of?

A

a SMRT cell with tens of thousands of zero-mode waveguides (ZMWs.)

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5
Q

What is the benefit of ZMWs?

A

ZMWs provide the smallest light detection volume ever (20 zeptoliters = 10-21L)

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6
Q

What light only can pass ZMW?

A

Only attenuated light can pass

The light illuminating the ZMW from below is of too high a wavelength to pass through the waveguide

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7
Q

How does SMRT cell sequencing work?

A

A DNA-polymerase complex is first immobilised to the ZMW bottom, followed by phosphorylated nucleotides being added to the ZMW chamber. The four different bases are labelled with four different fluorophores and a light pulse is produced with each base added within the chamber. The small detection volume minimises background noise 1000-fold, and this occurs across all the ZMWs on the SMRT cell simultaneously.

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8
Q

What does nanopore sequencing allow?

A

DNA bound to a processing enzyme to be read in real-time with increased flexibility

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9
Q

How is nanopore sequencing done?

A

The bases are sequenced using differential changes to the electrical field as the DNA is pulled through the nanopore. The speed of the enzyme can be modulated, and the sequencing run until the scientist requires – not based on arbitrary instrument run-times.

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10
Q

What could nanopore and SMRT sequencing be used for?

A

to sequence more complex genomes – such as those in agriculturally important plants – in order to better genome engineering projects in these organisms to facilitate human health.

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11
Q

What does single cell sequencing remove the requirement of?

A

A cell collection being used to obtain a large amount of DNA to sequence.

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12
Q

How does SCS emoves the requirement of a cell collection being used to obtain a large amount of DNA to sequence?

A

lysing a single cell in a microwell/droplet and adding an oligonucleotide sequence to act as a molecular barcode.

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13
Q

Following lysis of a cell in a microwell/droplet and adding an oligonucleotide sequence to act as a molecular barcode, what happens in SCS?

A

The DNA and RNA can be extracted and amplified using PCR.

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14
Q

What can be used to collapse PCR duplicates and compensate for non-uniform amplification?

A

Unique Molecular Identifiers (UMIs)

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15
Q

Single cell RNA-seq has been used to reveal what?

A

cellular hierarchies within tissues and previously undiscovered cell-types within organs

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16
Q

What technique can be applied to tumour biopsies in order to understand changes in tumour cells and the evolution of the tumour before and after treatment?

A

Single Cell RNA-seq

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17
Q

List the limitations of the CRISPR-Cas9 system

A

1) Specificity
2) Safety
3) Efficiency
4) Versatility

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18
Q

Explain how there are specificity concerns with CRISPR-Cas9

A

Cas9 could cut at off-target sites, even with a small number of mismatches

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19
Q

Explain how there are safety concerns with CRISPR-Cas9

A

On-target cuts could damage the genome in ways not yet understood:
Indels, knock-ins and large-scale insertions, deletions and translocations which depend on an alt-NHEJ pathway

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20
Q

Explain how ther are specifiicity concerns with CRISPR-Cas9

A

NHEJ greatly outcompetes HDR methods

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21
Q

Explain how there are versatility concerns with CRISPR-Cas9

A

Editing is restricted to target loci with a PAM sequence

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22
Q

How can specificity issues of CRISPR-Cas9 be addressed?

A

in silico methods to search for similar sequences to the gRNA which may be cut. The gRNA with the lowest similarity hit-rate can be selected. Factors influencing specificity include:

  • GC-content of the heteroduplex
  • Position of gRNA:DNA mismatches relative to the PAM
  • Chromatin status of targets
  • Scaffold RNA sequence
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23
Q

What is a current method under development to lower the probability of having an off-target effect using CRISPR-Cas9?

A

Utilising paired nickases to form DSBs, rather than if a full nuclease was used

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24
Q

How could you minimise the safety concerns involved with CRISPR-Cas9?

A
  • Identifying and temporarily suppressing alt-NHEJ components
  • Avoiding DSB generation would prove to be the safest method and can be achieved by fusing dCas9 to a base editor such as cytosine deaminase which can edit a CG pair to an AT pair.
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25
Q

How can the efficiency of CRISPR-Cas9 be improved?

A

By temporarily inhibiting NHEJ components, such as DNA-PK in order to channel DSBs into the HDR pathway as the NHEJ pathway is no longer competing.

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26
Q

How can versatility issues be overcome in the CRISPR-Cas9 system?

A

by using several CRISPR/Cas9 systems in addition to that of S. pyogens. The systems from other bacteria and archaea utilise different PAM sequences and cutting methods so one can be chosen based on convenience for the project at hand.

27
Q

What are the different types of CRISPR-Cas9 systems?

A

a) crRNA-RNA pairing
b) crRNA-DNA pairing
these can be either class 1 or class 2
Type 1-5
- Types I, II, and V generate an RNA-DNA hybrid, displacing the nontarget DNA strand. The target strand contains the protospacer (red), which is complementary to the spacer sequence in the guide RNA (orange). The protospacer-adjacent motif (PAM, blue) is located at either the 3′ end (types I and V) or the 5′ end (type II) of the protospacer.
- Types III and VI recognize RNA targets requiring, respectively, an RNA-based PAM (rPAM) or a protospacer-flanking sequence (PFS) motif.

28
Q

What does gene therapy have the potential to do?

A
  • to provide a once-off treatment for a wide-range of diseases through the delivery of a therapeutic gene to an appropriate cell type
  • treat acquired diseases such as viral infections and cancer
29
Q

How can efficient delivery in gene transfer be carried out?

A

using viral vectors

30
Q

Where does the viral vector need to reach in dividing and non-dividing cell in order to be stably expressed?

A

Dividing cells require genomic integration for stable expression whereas non-dividing cells require delivery into the nucleus, but not necessarily genomic integration.

31
Q

What are the safety concerns with gene therapy?

A

Immune response to the viral vectors or genomic damage.

32
Q

What was a recent success of gene therapy?

A

Treatment of boys with X-linked SCID

33
Q

Explain the gene therapy treatment of boys with SCID?

A

Their HSCs were infected with a viral vector carrying cDNA encoding IL2Rg – the protein missing in SCID. This integrated into the genome and allowed most patients to develop full T-cell immunity.

34
Q

What were the problems with gene therapy for patients with X-linked SCID?

A

However, some patients developed T-cell Leukaemia and one died. This was due to the virus integrating close to a protooncogene and its LTR contained enhancers which upregulated it.

35
Q

How have recet trials redced the problems with gene therapy for X-linked SCID?

A

Recent trials have therefore removed the LTR enhancers in the vectors.

36
Q

WWhat does targeted gene therapy do?

A

It utilises gene editing to correct or modify the existing genes in an individual’s cells.

37
Q

What are the advantages of targeted gene therapy?

A
  • Safety – reduced chance of oncogene activation
  • Efficacy – normal gene expression can be restored of a mutated gene
  • Versatility – applicable to dominant diseases
38
Q

The advent of what has made gene correction therapy a practical reality?

A

customised endonucleases

39
Q

What are the four types of gene editing that can be used for therapeutic benefit?

A
  • Gene disruption – pathogenic genes can be silenced
  • NHEJ correction – pathogenic insertions can be deleted
  • HDR correction – deleterious mutations can be corrected
  • HDR gene addition – therapeutic gene introduced
40
Q

How does NHEJ correction work?

A

DSB repaired through NHEJ creates an indel that results in a truncated or non-functional protein

41
Q

How does gene disruption work?

A

DSBs generated and repaired through NHEJ to make the protein functional again

42
Q

How does HDR correction work?

A

DSBs are generated in the mutated regions and HDR is used to integrate the corrected HDR template and form a corrected protein

43
Q

How does HDR gene addition work?

A

DSBs created in a ‘safe harbour locus’ and HDR repair can insert the therapeutic gene to produce a functional protein

44
Q

What is becoming an important tool in the feild of personalised medicine?

A

genomics

45
Q

What is the 100,000 genomes project?

A

The sequencing 100,000 genomes of patients with cancer and other rare conditions in order to increase the understanding of genetic variants, accelerating the integration of genomic medicine into the NHS, promote public understanding into the benefits of genomic medicine and stimulate the UK life sciences industry and commercial activity in genomics.

46
Q

What would be a potential problem of implementing the 100,000 genomes project in the NHS? What is being done to rectify that?

A

the insufficient training healthcare workers have in genomics – thus the Genomic Education Programme has been launched

47
Q

The study of what can help administer the correct drug at the correct dose for an individual to increase effectiveness whilst reducing adverse side effects?

A

Pharmacogenomics

48
Q

What genetic markers can be screened for prior to treatment to predict whether side effects will be experienced?

A

HLA-B*5701 or TPMT

49
Q

Why is personalised medicine and pharmacogenetics relevant to cancer treatment?

A

as every tumour is different and responds differently to treatments. Research is looking into which mutations affect drug-sensitivity and mutational combinations affecting drug-sensitivity.

50
Q

Give an example of why reaserch into which mutations affect drug-sensitivity and mutational combinations affecting drug-sensitivity, is not always successful?

A

Melanoma with mutations in the BRAF gene responds well to B-raf-inhibitors but then becomes resistant to the treatment

51
Q

What three factors must be understood to better approach to personalised medicine?

A
  • Risk – expected probability that a patient will develop a particular disease
  • Screening – testing seemingly healthy individuals to allow for early diagnosis
  • Prevention – lifestyle changes, drugs or other treatments employed to reduce the risk of disease development
52
Q

How are antibodies only containing human sequences, with a high affinity, engineered?

A

In vitro or in vivo by immunising mice transgenic (humanised mice) for human Ig genes.

53
Q

What are humanised pigs used for?

A

Xenotransplantation of organs to avoid immune responses and the addition of porcine endogenous retroviral genomes (PERV) which can infect a human.

54
Q

What must be done to make a humanised pig?

A

Several genes must be knocked-out to engineer a humanised pig, this has become possible with the advent of CRISPR gene editing techniques.

55
Q

What gene editing has been done to reduce the prevalence of malaria?

A

Gene-editing in mosquitoes to render them infertile or unable to host the Plasmodium to prevent malarial spread

56
Q

What is gene drive?

A

a process by which a modified chromosomal locus in germ cells can code for an endonuclease which cuts the unaltered locus. HR repair allows for the cut locus to become modified as the modified locus is used as a template, resulting in a germ cell homozygous for a modified locus. Gene drive spreads completely through a mosquito population within a couple of generations and CRISPR methods are being used for this application.

57
Q

What is the mechanism and population-level effect of endonuclease gene drives?
(explanation 2)

A

a) Endonucleases cut competing alleles, inducing the cell to repair the damage by copying the endonuclease gene.
(b) By converting heterozygous germline cells into homozygotes containing two copies, gene drives increase the odds that they will be inherited and consequently spread themselves and associated changes through wild populations.

58
Q

What is an important consideration when carrying out gene drive?

A

The process is irreversible, it must be done with utmost care and careful consideration.

59
Q

What is ageing hypothesised to be?

A

The accumulation of genomic damage to our somatic cells that cause the altered physiology associated with ageing

60
Q

What are the altered physiologies assocaited with aging?

Dylan Said Ceara Might Eat Trees And Grass

A 
Deregulated nutrient sensing 
Stem cell exhaustion 
Cellular senescence 
Mitochondrial dysfunction 
Epigenetic alterations 
Telomere attrition 
Altered intercellular communication 
Genomic instability
61
Q

What have been suggested as potential ways to slow aging?

A
  • Upregulating telomerase genes DKC1 and TERC1

- Mutations inhibiting IGF1 function have been shown to increase longevity

62
Q

What are the key ethical considerations that need to take place when dealing with applications of genetics and genomics?

A
  • Privacy of personal genome sequence data
  • Interpreting commercially generated personal genome sequences
    Somatic versus germ-line therapy
  • Genetically modified humans and ‘designer babies’
  • Environmental impacts of genetically modified organisms
  • Societal consequences of extended lifespans
63
Q

Give a valid definition of pharmacogenetics

A

The use of genome data to design new pharmaceuticals

Genetics (10 decks)

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