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Regulation Of Gene Expression > Lesson 11 > Flashcards

Lesson 11 Flashcards

1
Q

what are the three ways that sgRNAs and Cas9 proteins are delivered to cells?

A

viral or plasmid vectors, mRNA delivery, and protein delivery

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2
Q

describe the delivery of sgRNAs and Cas9:

A

can be delivered by the same or different vectors → can combine in target cells if they are encoded by two different vectors

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3
Q

what two vectors can be used as viral / plasmid vectors?

A

Adeno-associated virus (AAV) or lentiviruses

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4
Q

describe sgRNA and Cas9 delivery through mRNA delivery:

A

chemically synthesized in vitro and the preferred way to deliver mRNAs are nanoparticles

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5
Q

what is the most important thing for the treatment of genetic diseases using gene therapy?

A

to know which genes are the causative genes

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6
Q

what is the specificity of the CRISPR-Cas system dictated by?

A

the PAM sequence and the sequence of the 5’ of the guide RNA

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7
Q

up to how many mismatches can be tolerated between the bound target DNA and the sgRNA?

A

up to 5 (in some cases there isn’t a high specificity of recognition)

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8
Q

what cell line is often used for the study of gene therapy?

A

human pluripotent stem cells

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9
Q

why are iPSCs common?

A

some tissues it is very difficult to obtain primary cells for study (hepatocytes, brain cells, lung tissue) → they are easy to harvest and test in vitro

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10
Q

due to the strict regulations on human embryos, what is a source of similar embryo tissue?

A

tripronuclear zygotes

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11
Q

name three characteristics of tripronuclear zygotes:

A

-are constituted by 1 oocyte nucleus and 2 sperm nuclei.
-are discarded if they occur in nature.
-can be used for research and they are destroyed after the manipulations

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12
Q

where is gene addition and editing forbidden in regards to cell type?

A

germ-line cells

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13
Q

where is most gene therapy research performed?

A

somatic cell lines

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14
Q

in one of the first studies performed in order to investigate the specific editing and off-target effects with CRISPR/Cas, what target gene was used?

A

HBB - huma beta-globulin genes

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15
Q

using the HBB sequence, the group was able to design what to cover the three most common mutations?

A

three different single guide RNA

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16
Q

if there is a high rate of off-target modifications compared to the on target genes, what does this mean?

A

there is a problem

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17
Q

the repair template of HDR can be either of what two things?

A

the endogenous homologous gene or the exogenous DNA sequence

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18
Q

when studying hypertrophic cardiomyopathy, where was the CRISP/Cas system delivered?

A

delivered in the zygotes in S phase or M phase using a cytoplasmic sperm injection

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19
Q

how did scientist correct mutations found in hypertrophic cardiomyopathy?

A

provided a template to induce HDR to fix the mutations → corrected using th heathy gene from the egg genome (egg genome was used to fix mutation but this process was not expected because the preferential material was the template DNA injected
from outside)

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20
Q

what coreceptors does HIV need?

A

CD4+ and the CCR5 coreceptor to enter T cells

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21
Q

what was discovered in regards to the impairment of HIV?

A

if there is a mutation (delta 32 deletion in the exon) in the CCR5 gene that there will be an impairment when the HIV virus enters into the T cells

22
Q

the mutations and the implication of the delta32 in HIV patients were discovered how?

A

the Berlin patient → received the bone marrow and so, he became resistant to HIV infection and then was cured for the tumor and the AIDS

23
Q

what confirmed the results of the Berlin patient?

A

the London patient → showed the remission from HIV was possible after the HSC transplantation (bone marrow transplantation) which can replace the hematopoiesis system in the recipient)

24
Q

after the first CRISPR babies, what was set in place by the scientific community?

A

a moratorium which settled what can and can’t be done with the CRISPR system

24
Q

the CRISPR babies made what two points clear?

A

the system is a very potent, easy to use and efficient tool but general rules are needed → a balanced needs to be reached

24
Q

what did the NIH create last year?

A

the somatic cell genome editing program (SCGE)

24
Q

what is the function of the SCGE consortium?

A

the aim to accelerate the development of safer and more effective methods to edit the genome of somatic cells

24
Q

what are the three main types of editors?

A

genome editors, RNA editors, and epigenome editors

24
Q

what can genome editors be used for (such as CRISPR)?

A

editing both nuclear DNA and mtDNA

25
Q

what are epigenome editors?

A

the epigenome can be a molecular target of editors because the level of gene expression is important; can also edit mtDNA

26
Q

where can target tissues or organs be edited?

A

both ex-vivo and in-vivo

27
Q

what type of editing has received a lot of attention as potential to cure disease in low income countries?

A

in vivo editing of the somatic cell genome → easier, less expensive, more sustainable for countries with many patients and fewer institutions

28
Q

what other models are preferred for testing besides mice?

A

non-human primates and organoids

29
Q

what are base editors?

A

they can change a single base creating a single somatic mutation but not create INDEL

30
Q

why are base editors important?

A

many diseases are caused by just one single nucleotide mutation

31
Q

what method of delivery is actively being studied?

A

non-viral development of lipid-nanoparticles

32
Q

describe nanoparticles:

A

nanoparticles have a similar composition to the cellular membrane → therefore when nanoparticles are injected in vivo into the organism, they can enter in any type of cell

33
Q

what are the four ways different nanoparticles can be administered?

A

systemically, intranvenously, locally (directly into tissues), or ocularly

34
Q

name two facets of gene therapy:

A

gene addition and gene editing

35
Q

what are the only two diseases in which now there are results form both gene editing and gene addition?

A

Beta-thalassemia and SCD (sickle cell)

36
Q

what common defect is found in Beta-thalassemia and SCD (sickle cell)?

A

defect in hemoglobin synthesis

37
Q

where are the genes for the alpha strand of hemoglobin located?

A

chromosome 16 alpha

38
Q

where are the genes for the beta strand of hemoglobin located?

A

chromosome 11 beta

39
Q

what is the Alpha cluster composed of in hemoglobin?

A
  • ζ (zeta): present in the embryonic hemoglobin
  • α2: present the fetal and postnatal hemoglobin
  • α1: present the fetal and postnatal hemoglobin
40
Q

what is the beta cluster composed of in hemoglobin?

A
  • ε: encode for the embryonic chain of embryonic hemoglobin;
  • GγAγ: these two γ gene encodes for the γ chains, for the fetal hemoglobin;
  • δ and β: the adult gene
41
Q

describe hemoglobin during development:

A

During development, organisms use different forms of hemoglobin because the oxygenation of the tissues is different in the uterus and after birth

42
Q

the regulation of the alpha cluster is obtained by what?

A

regulatory element HS-40 (hypersensitivity site 40) and the single gene promotor

43
Q

how is the beta cluster regulated?

A

BLCR and five different hypersensitivity sites

44
Q

when is the embryonic form of hemoglobin present?

A

during the first trimester

45
Q

where do the switches between the different states occur?

A

the first switch occurs in the Yolk sac, while the second one is in the fetal liver (in mice and in men the hematopoietic organ during fetal life is the liver) around birth, all the cells migrate into the bone marrow and colonize it

46
Q
A

Regulation Of Gene Expression (28 decks)

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