Lecture 13 Flashcards
What are the ways genetic information is used in medicine?
- Actionable
- Can help inform on clinical decisions
- Deterministic
- Condemnation
- Being aware of why we are doing these things like questioning the ethics on gene editing
- Condemnation
- Probabilistic:
- Increased “risk”
- Reason: disease might only affect small % of population so some might consider it “useless”
- Increased “risk”
Distinguish between ex vivo, in vivo, and in vitro
Ex vivo:
- Form of Somatic editing therapy
- Individual’s cells are removed, edited, and then reintroduced
In vivo: viral delivery of genes
- Form of Somatic editing therapy
- Use of viral plasmids that are transfected into packing cells on media to produce viral vectors which can be injected into the individual
* Before putting the cells back into the body, genetic testing can be done on the viral vectors to identify on- and off-target effects
- off- target effects refer to changes or other effects in the genome that were not purposely intended. But whenever we change things in the genome we have that possibility to introduce other mutations or structural variants into the genome.
In vitro:
- Form of germline editing medicine
*Cells are removed via IVF, edited in the embryo stage, on- and off- target analyses , and reintroduce verified blastocysts via intrauterine transfer
Describe the molecular principle of CRISPR genome editing
*Using Cas9 endonuclease:
- Cas9 + sgRNA (single-guide RNA)
- sgRNA is a synthetic RNA made of 2 RNAs in a bacteriophage : crRNA + tracrRNA
1) The RNA forms a 2° structure - the stem loop – Cas9 binds to this structure
2) sgRNA is made of a sequence that matches the target DNA. It forms a base pairing interaction the target DNA, telling Cas9 enzyme WHERE to cut – makes a DSB by making 2 cuts (ends up inly being 1 DSB at the end)
- PAM (NGG) is a sequence immediately downstream of the target DNA that allows the Cas9-sgRNA complex to bind
3) NHEJ OR HDR for repair of DSB
Describe Homologous Directed Repair (HDR)
- form of homologous recombination but instead of recombining off of a homologous chr. you introduce a short segment of what you want to edit + replace
What are some outcomes of NHEJ?
Can happen to try to repair the DSB but:
- is error prone
- can form indels (can cause frameshift/Nonsense –> premature STOP codon)
Describe Cas12
- Newer and more precise for gene editing
- See less off-target effects
- Makes staggered DSBs which means that they are offset from each other and so you have a little bit of ssDNA at the tails –> which favors HDR
- It has a different PAM (TTTV instead of NGG) sequence so it is good at editing regions that are A/T rich
What are the differences between dCas9 and Cas9?
*dCas9 = deadCas9
- can still bind to sgRNA + target and localized specific mutations in the genome BUT DOES NOT CUT DNA
- Important because we can make gene fusion with CRISPR and some sort of protein that can do something at a specific genomic location
Ex: making fusion with regions that make transcription activation proteins
- Like dCas9 KRAB-MeCP2 or dCas9-HDAC1
- Point is, we do NOT have to cut the DNA to have some effect, can just deliver a protein to a region of a gene to have some effect
*Cas9 on the other hand can cut DNA
Describe base editing
Ex: Using deaminases – are enzymes can just go in and modify a base to create a specific mutation
What are the different ways one can use dCas9?
1) Creating Gene fusions:
a) Transcription activating:
- A gene fusion of dCas9 + activation domain resulting in a protein that helps to recruit RNA polymerase to the site of the promoter + activate txn
b) Transcription repressing:
- A gene fusion of dCas9 + Interference domain that prevents RNA pol from being recruited to this region so transcription cannot be initiated
2) Creating epigenetic marks
a) Histone acetyltransferase (HAT)(dCas9-P300)
- Introducting dCas9 so that cells can modifying histone proteins of nucleosomes as a post-translational modification – changes how compact nucleosome
- Helps to activate txn
b) DNA methyltransferase (dCas9-DNMT3):
- Methylate cytosines that are in front of th promoter
- silences txn of a particular gene
What are LNPs?
Lipid nanoparticles:
- These particles help to stabilize RNA so they can get introduced without being immediately degraded by the individual’s body cells.
- The LNP allows the Cas9 + mRNA + chemically modified sgRNAs to be packaged within the LNP and can fuse with cells due to its lipid membrane
Describe Sickle Cell Disease
- First described in 1910
- It is caused by a mutation in the β-globin gene
- Mutated β-globin gene –> creates a mutated β-globin protein that is misfolded and forms fibrils –> the fibrils cause the sickle-shaped deformation - Affects 100,000 Americans which is considered rare (<200k)
- There are 2 FDA approved gene therapies for this disease since 2023
- Cost is $2.2 -3.1 million
- Gene editing with CRISPR by in 2 ways: edit based or KO repressor of fHb
Explain the ways to use CRISPR to treat sickle cell disease
*Using gene editing with CRISPR, 2 ways:
1) Edit based
- Using guideRNA + Cas9, can target and repair β-globin gene by correcting the incorrect base using ex-vivo methods for the individual’s body to start producing Normal RBCs
2) KO (knockout) repressor of fetal globin
- Cas9 promotes the production of fetal hemoglobin by breaking the gene that encodes for a repressor for fHb like BCL11A
- By knocking out the BCL11A gene, fHb cannot be repressed and can be expressed to prevent RBCs from sickling
What are the risks of CRISPR and possible solutions?
- Can have off-target effects – which are effects that are unintended consequences
- Cas12a has FEWER off-target effects
What are the 2 ways of in-vivo somatic editing:
- Lentivirus (Based on HIV):
- Take lentivirus –> modify it to get into nucleus –> get reverse txn from ints RNA genome –> get DNA using RT 2X for dsDNA –> dsDNA will integrate into the genome
- Components of the vector for lentiviruses:
a) integrating (Can possible integrate into important region of genome and affect expression/fxn of another gene)
b) Long lasting
c) High level of expression (pro)
- Adeno-associated virus (AAV):
- A DNA virus that does NOT integrate into the genome (this is an advantage, but sometimes they can)
- This AAV forms a circular DNA that can replicated in infected cells which will keep expressing the trans gene infected
- Components of the vector for AAV:
a) Non-integrating for the most part
b) High expression
c) Long term effect but not forever (up to a year or so)
