Unit 5: Gene Editing And CRISPR Flashcards
*What is gene editing?
Modifications directed at the genome, their contexts (epigenetic marks) or their results (transcripts) using endonuclease
Endonuclease=enzyme that cuts DNA
*What was the previous technology used for gene editing?
Recombinant DNA
*What gene editing tools do we use now?
- Zinc Finger Nucleases (ZFNs): High cost + difficulty, operating problems
- Transcription Activator-Like Effector Nucleases (TALENs): More difficult + expensive than CRISPR
- CRISPR-Cas
*What is CRISPR-Cas?
- It is a natural defence system against viruses in bacteria + archaea
- CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats
- Cas: CRISPR-associated
What are the stages of the natural system CRISPR-Cas was found in?
Accquisition, expression + interference
See diagram of how/where pg.6
Who was the Spanish scientist involved in the discovery of CRISPR?
Francisco Martínez Mojica
*Who won the Nobel Prize in Chemistry in 2020?
- Emmanuelle Charpentier + Jennifer A. Doudna
- “for the development of a method of genome editing
Who did Charpentier + Doudna fight over ownership of use of CRISPR-Cas with?
-Feng Zhang
*What is Germinal genetic modification?
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
When was research on use of CRISPR-Cas in germinal genetic genetic modification published?
- From 2015 onwards
- only 12 so far all from 2015-2019
- 2015 x1, 2016 x2, 2017 x5, 2018 x3, 2019 x3
*What are DNA repair mechanisms?
- Non-homologous end- joining (NHEJ)
- Homology- directed repair (HDR)
What are the risks of germinal gene editing?
- 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:
*Quotes on risks (from 2015 research paper)
- “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”
*What were the 3 research papers on CRISPR-Cas use in human embryos from 2020 on?
- *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
*How many human embryos were destroyed in 10 papers being published?
At least 866 human embryos destroyed (probably more)
*What are the ethical issues of genetic modifications on human embryos?
- 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
*In which cases is there no alternative for parents?
- 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
What is an autosomal dominant disease?
If one parent carries 2 disease-causing alleles (homozygous affected), all children will inherit the disease-causing genotype
What is an autosomal recessive disease?
If both parents carry 2 disease-causing alleles in the same gene (homozygous affected), all all children will inherit the disease-causing genotype
What are X-linked recessive diseases?
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
What was the Jiankui case?
- 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
What were the Jiankui case specifications?
- 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
*What is the current position of the scientific community?
- *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
*What is the trend seen in scientists from Europe?
- *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
*What was the statement from the 2nd international summit on human genome editing
“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)
What did the National Academy of Medicine, National Academy of Sciences and the Royal Society say about germline editing?
“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 ”
What did Valencian researcher Bellver Capella say about editing risks?
- 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
What is somatic gene modification?
- 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
*How can aggressive leukaemia be treated with somatic gene modifications?
- Experimental treatment based on *TALENs on the so-called *UCART19 cells
*What is Hunter Syndrome?
The *inability of the body to breakdown + recycle some sugars, due to deficiency of enzyme iduronate-2-sulfatase
*How can Hunter syndrome be treated?
- Using ZFN
- results not as promising as trial
What other disease is going through trials for somatic gene modification + why is it a good choice?
- Leber congenital amaurosis 10 (LCA10) which causes blindness
- Safer: working in eye safer than other organs
- Blindness not terminal, can be lived with
*What are somatic genetic editing ethical issues?
- Biosafety hazards:
- Off-target effects
- Immune response to Cas9
- Relationship with cancer
- Controlled clinical trials
- Proper disease selection
**Examples of CRISPR-edited plants
MUST READ
- Soybean (Glycine max) with drought + salt tolerance: achieved by disrupting Drb2a + Drb2b genes (double stranded RNA binding protein2 genes)
- Camelina with increase oil content: target genes not disclosed
- Setaria viridis/green bristle grass with delayed flowering time: deactivate S. viridis homologous of Zea mays ID1 gene
- Waxy corn with exclusively amylopectin starch: Inactivate endogenous waxy gene Wx1 that encodes granule-bound start synthase catalysing amylose production
- White button mushroom (agarics biporus) with antibrowning properties: Gene coding for polyphenol oxidase (PPO) knocked out
Genetic editing + consumption ethical issues?
- Do gene-edited foods deserve looser regulation than transgenic foods?: Traditional mutagenesis + hybridisation => Transgenic organisms => Gene edited. (Least to most)
Transgenic organisms vs. Gene editing
- 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
IDK WHAT TO TITLE THIS AS BUT LEARN*
- *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
