Ferrari: Lecture XI Flashcards
Genome Editing: Applications of a Powerful Tool
What are the editing tools of the CRIPSR/Cas9 system?
Cas9 protein and gRNA in the presence of sgRNA
Cas9 protein needs to be in the cells or tissues we want to edit
What 3 ways can use the CRISPR/Cas9 delivery system?
viral or plasmid vectors
mRNA delivery
protein delivery
Describe the delivery of CRISPR/Cas9 through viral or plasmid vectors:
the sgRNA and the Cas9 proteins are delivered by the same or 2 different vectors
protein and RNA can combine in target cells if they are encoded by 2 different vectors
vectors can derive from Adeno-associated (AAV) or lentiviruses (most powerful tools used for cell editing)
Describe the delivery of CRISPR/Cas9 through mRNA delivery:
the Cas9 mRNA and the sgRNA are chemically synthesized in vitro and the preferred way to deliver mRNA s are through nanoparticles, which can be targeted to specific cells
the mRNA encodes for Cas9
sgRNA are modified
Describe the delivery of CRISPR/Ca9 through protein delivery:
used to deliver Cas9 and sgRNA
What is an example of monogenic diseases that can be treated with gene editing?
diseases that are rare, and in some cases severe
cystic fibrosis (CF)
Huntington’s Disease
Cardiomyopathies
SMA
β-thalassemia
Describe cycstic fibrosis (CF)
severe autosomal recessive disease with no causable treatment except for lung transplant
causative agent is CFTR and the tissue that can be targeted uing gene editing is in the lungs
frequency: 1/2500 individuals
What is an important thing for the treatment of genetic diseases using gene therapy?
known what are the causative agents
What are some considerations to take when using the CRISPR/Cas9 system?
specificity of CRISPR/Cas-9 is dictated by the PAM sequence
a lot of work was done in cancer cell lines
we can have an unintended mutation
human pluripotent stem cell is another cell lineage that can be used for studies
low-rate of off-target effects were detected in studies of mice-zygotes
Why are tripronuclear zygotes used?
there are limitations as to what can be done with human embryos, so a similar embryos tissue is used
What is 3PN?
a kind of in vitro fertilization product
Describe 3PNs:
1 oocyte nucleus and 2 sperm nuclei
they are discarded if they occur in nature
can be used for research and they are destroyed after the manipulations
Can gene addition and gene editing be done on germ-line cells?
NO because if the organism is fertile, the modifications on the germ line can be transmitted to offspring
What cells can gene therapy and gene editing be done on?
somatic cell lines ONLY
A Chinese group studied CRISPR work in human cells. Since human embryos cannot be used, what did they use?
tripronuclear zygotes
The chinese group used tripronuclear zygotes to investigate _____
specific editing and the off-target activity of the CRISPR/Cas system
What was the target gene investigated by the Chinese group?
HBB (human beta-globin gene)
What is TOPO cloning?
a molecular biology technique in which DNA fragments are cloned to specific vectors without the requirement for DNA ligases
What can in silico analysis help us predict?
off-target cleavage in the analysed samples or in the whole genome
What does HBB gene encode?
beta-globin
What is the target site of CRISPR/Cas9?
HBB
Why would there be a rate of 100% INDELs in HBB?
HBB is the target site of CRISPR/Cas9
What were the conclusions of the Chinese experiment?
system can cleave the endogenous gene efficiently
The repair gene of HDR can be either ___ or ___.
endogenous homologous gene
exogenous DNA sequence
Is there a possibility to have spontaneous HDR even if we don’t provide the DNA template?
yes
What characterizes hypertrophic cardiopyopathy?
hypertrophy of the right ventricle
myofibrillar disarray
myocardial stiffness leading to heart failure
What kind of mutation is present in hypertrophic cardiomyopathy?
dominant mutation
What is the biological material used to analyze hypertrophic cardiomyopathy?
oocytes and sperm
When is the CRISPR/Cas9 system delivered in the zygotes?
Sp phase or M phase using a cytoplasmic sperm injection
What does HIV need to enter T cells?
coreceptors
CD4+: surface marker expressed on T cells
CCR5 coreceptor
What mutation causes the impairment of the HIV viruses entering the T cells?
mutation (delta32 deletion in exon) in the CCR5 gene
If a patient has a homozygous delta32 mutation, what can they confer?
resistance to HIV infection
Why is the Berlin patient important and who is the Berlin patient?
Berlin patient is important because it helped us discover the implication of delta32 in HIV patients
the patient is someone who was treated for HIV and leukemia
the treatment for leukemia was bone marrow transplantation but the donor had a delta32 mutation
when the patient received the bone marrow, he become resistant to HIV infection and cured from the tumor and AIDS
Who is the London patient?
a patient who confirmed the same results found in the Berlin patient
Is HIV a genetic disease?
no but we can think to treat it with a genetic approach
Why did China want to create CRISPR babies?
they wanted to use the CRISPR/Cas9 system to induce mutations in the CCR5 and then create babies resistant to HIV
What is the moratorium of CRISPR a result of?
the scientific community being very upset with the CRISPR babies
the moratorium set the guideline about what can and cannot be done with CRISPR, and it is effective in all laboratories
Name 2 points learned from the 3 CRISPR babies known to have been created:
CRISPR/Cas9 system is a very potent tool, easy to use, and efficient: however, rules should always be established and everyone should follow to avoid unpleasant outcomes
the key is to find balance in genome editing for future therapies
What is the SCGE consortium?
created by NIH; it aims to accelerate the development of safer and more effective methods to edit the genome of somatic cells from patients, even in tissues that are difficult to reach
What is the goal of NIH in support of the SCGE Consortium?
to cure diseases
Name and describe the 3 types of genome editors:
genome editors: such as CRISPR/Cas9 & they can be used to edit nuclear DNA and mtDNA
RNA editors: newer technology
Epigenome editors: molecular target of editors because the level of expression is important (can also edit mitochondrial DNA)
How can the target tissue or organs be edited?
both in-vivo or ex-vivo (if tissue is extracted from patient)
Which type of genome editing is easier: in-vivo or ex-vivo?
in-vivo and it is less expensive and more sustainable for countries with many patients and few institutions
What needs to be done to ensure the delivery of genome editing?
delivery tools closer to the resemblance of humans (such as non-human primates and organoids) must be developed
Name some new editors that have been introduced that are alternatives for TALEN, ZFN, & CRISPR/Cas9:
gene disruption
gene correction
epigenetic modification
off-target
mitochondrial editing
What is a base editor?
change a single base and create a single somatic mutation but not create INDEL
What is important to remember about base editors?
they are efficient, but it is important to always check the off-target because this could lead to something going wrong if we change a base that is not the targeted one
What are nanoparticles and why should we create specific nanoparticles?
nanoparticles are particles that can enter any cell and they have been engineered with something that drives them to the right cell/tissue
What are 4 ways nanoparticles can be delivered?
systemically
intravenously: to reach tissues quickly
locally: muscles
ocular: ocular injection
What are some canidate diseases for gene therapy (by gene addition and gene editing)?
Hemoglobinopathies
β-thalessemia
SCD
Define gene addition
addition of a gene to a genome
What is exciting about gene editing recently?
human results are very recent, but they are promising
What are the 2 diseases in which there are now results from gene addition and gene editing?
β-thalessemia and SCD (sickle cell disease)
Where is the defect in β-thalessemia and SCD?
hemoglobin synthesis
What is hemoglobin composed of?
2𝛼 and 2β chains
Where are the genes for the the 2 chains of hemoglobin located?
in 2 different chromosomes (chromosome 16 (𝛼) and chromosome 11 (β))
What 3 genes compose the 𝛼 cluster?
ζ (zeta): present in embryonic hemoglobin
𝛼₂: present in natal and postnatal hemoglobin
𝛼₁: present in natal and postnatal hemoglobin
What genes compose the β cluster?
ε: encode for the embryonic chain of embryonic hemoglobin
GᵧAᵧ: these 2 code for the gamma chains of the fetal hemoglobin
δ and β: adult gene
What is neat about the hemoglobin?
there are different forms of hemoglobin
What do the different forms of hemoglobin depend on?
oxygenation of tissues since it is different in the uterus and at birth
What percent of adult hemoglobin (HbA) is made from 𝛼 and β chains?
more than 90%
What percent of adult hemoglobin (HbA) is made from δ chains?
a fraction, and it is called HbA₂
What is the function of hypersensitive site 40 (HS-40) and the single gene promotor?
regulates the 𝛼 cluster
Define gene:
coding region + promoter
Describe the image:
embryonic form is during the 1st trimester
𝜸 globulin chain: the embryonic chains no longer are expressed and the 𝜸 genes are expressed at their maximum during their fetal life
at birth (δ & β): 𝜸 genes are repressed and completely shut off
What are the cross points called?
switch points
Where do all the cellular processes of the hemoglobin take place?
erythropoietic progenitor
Where does the first switch of hemoglobin chains occur?
yolk sac
Where does the second switch of hemoglobin chains occur?
fetal liver
Where does the third switch of hemoglobin chains occur?
bone marrow and the cells colonize it
