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Biology > Unit 5: Gene Editing And CRISPR > Flashcards

Unit 5: Gene Editing And CRISPR Flashcards

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1
Q

*What is gene editing?

A

Modifications directed at the genome, their contexts (epigenetic marks) or their results (transcripts) using endonuclease

Endonuclease=enzyme that cuts DNA

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2
Q

*What was the previous technology used for gene editing?

A

Recombinant DNA

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3
Q

*What gene editing tools do we use now?

A
  • Zinc Finger Nucleases (ZFNs): High cost + difficulty, operating problems
  • Transcription Activator-Like Effector Nucleases (TALENs): More difficult + expensive than CRISPR
  • CRISPR-Cas
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4
Q

*What is CRISPR-Cas?

A
  • It is a natural defence system against viruses in bacteria + archaea
  • CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas: CRISPR-associated
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5
Q

What are the stages of the natural system CRISPR-Cas was found in?

A

Accquisition, expression + interference

See diagram of how/where pg.6

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6
Q

Who was the Spanish scientist involved in the discovery of CRISPR?

A

Francisco Martínez Mojica

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7
Q

*Who won the Nobel Prize in Chemistry in 2020?

A
  • Emmanuelle Charpentier + Jennifer A. Doudna

- “for the development of a method of genome editing

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8
Q

Who did Charpentier + Doudna fight over ownership of use of CRISPR-Cas with?

A

-Feng Zhang

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9
Q

*What is Germinal genetic modification?

A

In *reproductive cells (sperm + egg) are modified by introducing functional genes into their genome or by disrupting the wrong genes
- Takes place in *early embryonic development

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10
Q

When was research on use of CRISPR-Cas in germinal genetic genetic modification published?

A
  • From 2015 onwards
  • only 12 so far all from 2015-2019
  • 2015 x1, 2016 x2, 2017 x5, 2018 x3, 2019 x3
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11
Q

*What are DNA repair mechanisms?

A
  • Non-homologous end- joining (NHEJ)

- Homology- directed repair (HDR)

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12
Q

What are the risks of germinal gene editing?

A
  • Off-target effects: modifications that occur in another area (uncontrolled)
  • Repair by NHEJ route: random insertions or deletions (mutations on target, low efficiency HDR route)
  • Mosaicism:
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13
Q

*Quotes on risks (from 2015 research paper)

A
    • “Because the gene edited embryos are genetically mosaic, it would be impossible to predict gene editing outcomes through pre-implantation genetic diagnosis (PGD)”
    • the pressing need to further improve the fidelity and specificity”
    • “computational prediction based on sequence similarity to target sites failed to predict many off-target mutations”
    • “not a current option due to both ethical and technical issues”
    • “specificity of base editors needs more comprehensive investigation”
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14
Q

*What were the 3 research papers on CRISPR-Cas use in human embryos from 2020 on?

A
  • *Frequent loss-of-heterozygosity in CRISPR-Cas-9-edited early human embryos
  • *Allele-Specific Chromosomal Removal after Cas9 cleavage in human embryos
  • *Frequent gene conversion in human embryos induced by double strand breaks
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15
Q

*How many human embryos were destroyed in 10 papers being published?

A

At least 866 human embryos destroyed (probably more)

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16
Q

*What are the ethical issues of genetic modifications on human embryos?

A
  • Informed consent: inability of embryo to give it, will affect all its offspring, will be subject of research
  • Therapy or reproductive option?: treatment foreseen before the conception of “patient”, questions about allocation of public resources
  • Lead to less inclusive society + discrimination?: Private exploitation of techniques can lead to higher + lower genetic levels. “Genetic imperfection” would be choice of “irresponsible” parents (alr. happening in Spain e.g Down’s Syndrome)
  • Ethical to produce “designer” children?: Germline gene editing opens door to human genetic improvement (case of children/ twins from China)
  • Association with IVF
17
Q

*In which cases is there no alternative for parents?

A
  • 4-8 x 10^-8% of couples could benefit from gene editing of a certain gene
  • cases of severe monogenic diseases in which all children would inherit the genotype of the disease
18
Q

What is an autosomal dominant disease?

A

If one parent carries 2 disease-causing alleles (homozygous affected), all children will inherit the disease-causing genotype

19
Q

What is an autosomal recessive disease?

A

If both parents carry 2 disease-causing alleles in the same gene (homozygous affected), all all children will inherit the disease-causing genotype

20
Q

What are X-linked recessive diseases?

A

If the expectant mother carries 2 disease-causing alleles (homozygous affected) + father carries 1 disease-causing allele on his only X chromosome (hemizygous affected), all offspring would be affected

21
Q

What was the Jiankui case?

A
  • 2 twin girls born in 2018, whose DNA was modified with CRISPR/Cas to give them “protect” them against HIV
  • Research has not been published
  • Scientists agree it was irresponsible
22
Q

What were the Jiankui case specifications?

A
  • Falsified ethical approval documents
  • Unfavourable benefit risk balance:
    - chose target disease that can be prevented + treated
    - Chose CCR5 gene which has important functions (still not fully known
    - Experiment not considered therapeutic, it’s “improvement”
  • Embryonic destruction
23
Q

*What is the current position of the scientific community?

A
  • *Declared that in the future the clinical application of germline genetic edditing could become a realistic option
  • *Door is not closed to a possible future use of germline gene therapy, but it is pointed out that today the unresolved issues make it innapropriate in embryos for later implantation
  • *Requested continuation of research on embryos but failure to carry out any reproductive application
24
Q

*What is the trend seen in scientists from Europe?

A
  • *In March 2016 representative from more than 20 European countries came together to reflect on + promote responsible research with CRISPR-Cas. In July 2017, the consensus document was published which reflected this vision
  • *Recommends conducting careful scientific research to built an evidence base
25
Q

*What was the statement from the 2nd international summit on human genome editing

A

“Progress over the last 3 years and the discussions at the current summit, however, *suggest that it is time to define a rigorous, responsible translational pathway toward such trials”

(first paper 2015, summit 2018)

26
Q

What did the National Academy of Medicine, National Academy of Sciences and the Royal Society say about germline editing?

A

“Human embryos whose genomes have been edited should not be used to create a pregnancy until it is established that precise genomic changed can be made reliably and without introducing undesired changes - criteria that has not been yet been met ”

27
Q

What did Valencian researcher Bellver Capella say about editing risks?

A
  • It is about *preventing research that produced serious unwanted effects and, with it, a social alarm that blocks research in promising field
  • *do not jeopardise the public’s confidence in those investigations. They do not think about the protection of the human embryo
28
Q

What is somatic gene modification?

A
  • The ability to introduce genetic material (RNA) into an appropriate cell type or tissue in vivo
  • *Safer than CRISPR/Cas: can analyse result before putting in
  • *Clinical trials are few but have already started, especially ex vivo trials

See diagram pg.45 for method

29
Q

*How can aggressive leukaemia be treated with somatic gene modifications?

A
  • Experimental treatment based on *TALENs on the so-called *UCART19 cells
30
Q

*What is Hunter Syndrome?

A

The *inability of the body to breakdown + recycle some sugars, due to deficiency of enzyme iduronate-2-sulfatase

31
Q

*How can Hunter syndrome be treated?

A
  • Using ZFN

- results not as promising as trial

32
Q

What other disease is going through trials for somatic gene modification + why is it a good choice?

A
  • Leber congenital amaurosis 10 (LCA10) which causes blindness
  • Safer: working in eye safer than other organs
  • Blindness not terminal, can be lived with
33
Q

*What are somatic genetic editing ethical issues?

A
  • Biosafety hazards:
    • Off-target effects
    • Immune response to Cas9
    • Relationship with cancer
  • Controlled clinical trials
  • Proper disease selection
34
Q

**Examples of CRISPR-edited plants

MUST READ

A
  1. Soybean (Glycine max) with drought + salt tolerance: achieved by disrupting Drb2a + Drb2b genes (double stranded RNA binding protein2 genes)
  2. Camelina with increase oil content: target genes not disclosed
  3. Setaria viridis/green bristle grass with delayed flowering time: deactivate S. viridis homologous of Zea mays ID1 gene
  4. Waxy corn with exclusively amylopectin starch: Inactivate endogenous waxy gene Wx1 that encodes granule-bound start synthase catalysing amylose production
  5. White button mushroom (agarics biporus) with antibrowning properties: Gene coding for polyphenol oxidase (PPO) knocked out
35
Q

Genetic editing + consumption ethical issues?

A
  • Do gene-edited foods deserve looser regulation than transgenic foods?: Traditional mutagenesis + hybridisation => Transgenic organisms => Gene edited. (Least to most)
36
Q

Transgenic organisms vs. Gene editing

A
  • GMO: contain foreign genes randomly introduced into genome that produce new proteins in the organism, giving a desirable trait not previously had
  • Gene editing: contain small alterations in existing genes giving beneficial trait by tweaking levels of a protein already in body
37
Q

IDK WHAT TO TITLE THIS AS BUT LEARN*

A
  • *Unwanted effects will occur les often in gene-edited products, potentially safer than traditional random mutagenesis products
  • *Characteristics of final product must be examined regardless of technique used
  • *Current approach does not adequately respect motivation behind precautionary principles of ensuring product safety
  • *Situations may arise where 2 identical products would have to meet different regulatory requirements, due to different method used in production

Biology (11 decks)

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