1/27/20:

We talked to Daniel about doing research on CRISPR (background, what it has done today, basic mechanics) we also made a schedule

2/3/20:

Have to have research done (shared notes in document)

RESEARCH:

https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/questions-and-answers-about-crispr

• CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats
• The program targets specific stretches of genetic code and allows for DNA editing in certain locations. This means it could modify genes and correct mutations or a genetic trait that causes a disease
• It was first discovered by Franciso Mojica, a scientist at the University of Alicante in Spain. His original idea of it was to serve as a part of the bacterial immune system, defending against invading viruses.
• Then in 2013, Zhang Lab published the first method to engineer CRISPR to edit the genome
• How it works:

• The CRISPR sequences are transcribed into short RNA sequences (CRISPR RNA’s or crRNA’s). When the DNA is targeted, Cas9 (one of the enzymes produced by CRISPR) binds to the DNA and cuts it, shutting the targeted gene off.

• {esentially, CRISPR has this thing inside it that cuts the introns in half and created it’s own malfunction to create a new gene/modify it?}

• What are the differences between this and other genome editing tools?

• CRISPR-Cas9 is efficient and customizable
• It does not need to be paired with separate cleaving enzymes (Restriction enzyme, a protein produced by bacteria that cleaves DNA at specific sites along the molecule. In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating infecting organisms.)

• Different ones:

• CRISPR-Cpf1 →

• It requires only a single RNA and the enzyme is also smaller, making it easier to deliver into cells and tissues.
• It also cuts far away from the site, meaning that even if the targeted gene gets mutated at the cut site, it can be re-cut to allow the opportunity for correct editing.
• Provides flexibility in choosing target site… must first attach to the short sequences (PAM)

• CRISPR-Cas9 → Requires both RNA strands and cuts both strands at the same place, leaving ‘blunt ends’ that often undergo mutations once they rejoin.

• CRISPR is now being developed as a rapid diagnostic

• In eukaryotic cells, RNA polymerase attaches to the promotor and makes a copy of DNA –> mRNA. mRNA is translated to protein in the ribosomes.
• 5’ A C C T A A T 3’

3’ T G G A T T A 5’

3’ A C C _ _ _ A T 5’

5’ T G G _ _ _ T A 3’

WHAT IT’S DONE SO FAR: https://futurism.com/neoscope/11-incredible-things-crispr-has-helped-us-achieve-in-2017

• Successful application of gene-editing human embryos in the US were confirmed by a research paper published in Nature. The researchers “corrected” one-cell embryo DNA to remove the MYBPC3 gene — known to cause hypertrophic cardiomyopathy (HCM), a heart disease that affects 1 in 500 people.
• successfully used gene editing to completely extract HIV from a living organism, with repeated success across three different animal models. The team also prevented the progress of acute latent infection.
• “Semi-synthetic organisms were developed by breeding E.coli bacteria with an anomalous six-letter genetic code, instead of the normal four-base sequence. Additional gene editing was implemented to ensure that the new DNA molecules were not identified as an invasive presence”
• successfully targeted the “command center” of cancer — called the hybrid fusion — which leads to abnormal tumor growths.
• Scientists also slowed the growth of cancerous cells, by targeting Tudor-SN, a key protein in cell division. They then used this technique to use to slow the growth of fast-growing cells.
• gene-editing techniques have also made superbugs kill themselves. By adding antibiotic-resistant gene sequences into bacteriophage viruses, self-destructive mechanisms are triggered which protect bacteria.
• mosquito-borne diseases an extinct phenomenon. By editing fertility genes, scientists had the ability to limit the spread of mosquitoes
• edited out Huntington’s disease from mice, pushing the symptomatic progression of the condition into reverse.
• provided a more abundant and sustainable biofuel. By connecting several gene-editing tools, scientists engineered algae that produce twice the biofuel material as wild (or “natural”) counterparts.

Why crispr is such a revolutionary advancement: https://www.jax.org/news-and-insights/jax-blog/2014/march/pros-and-cons-of-znfs-talens-and-crispr-cas

• TALEN and ZNF are both from the 1990s to now, they are the “older” tools used for gene editing

• Zinc finger proteins (ZNFs) were first, Transcription activator-like effector nucleases (TALENs) were second

• Both are effective but CRISPR/cas-9 system is better because:

• Its specificity and “simplicity” in location services

• 2 ZNF needed to be guided to either side of the location on DNA, plus it looks for specific amino acids, meaning triplets (nucleotides), TALENs can recognize individual nucleotides, but still pretty similar to ZNF
• Since it has guide RNA CRISPR/cas is much easier to program

• It is way more efficient in terms of actually editing
• “Mutations can be introduced in multiple genes at the same time by injecting them with multiple gRNAs.”
• All of them have disadvantages though:

• Off-site effects
• Mosaicism
• Multiple alleles

• https://www.youtube.com/watch?v=1BXYSGepx7Q

• “Quick” and “easy are relative terms, meaning not for the general public, but certainly helps those in possession of a lab

• Most commonly mentioned: Cystic fibrosis, sickle cell, huntington’s, muscular dystrophy, Cancer! (mostly in the blood eg leukemia, and they not editing the cancer cells the editing the tcells that are a part of ur immune system, talk more on that later) → single gene disorders are better tho → choose one

Cystic Fibrosis: https://ghr.nlm.nih.gov/gene/CFTR#conditions

• only one in 2,500 people are born with cystic fibrosis
• Caused by a mutation in the CFTR gene, most commonly in delta F508 (?), but there are other mutation locations that cause it
• The CFTR gene controls the production of the cystic fibrosis transmembrane conductance regulator protein, which controls the fluidity of the mucus in your lungs
• Essentially, this mutation causes mucus in your body to be super thick, affecting your lungs and blocking airways and causing damage along with the creation of scar tissue
• It can also affect digestive system, since mucus appears here too
• Current treatment:

• “Several therapies including mucolytics, inhaled antimicrobials, systemic anti-inflammatories, and nutritional support are the mainstays of CF treatment, and these supportive therapies are largely responsible for the marked improvement in life expectancy over time.” (link), essentially, there’s no cure, just treat the effects and see how that goes.
• Ivacaftor and Lumacaftor, both drugs that actively interact with the protein and alleviate effects

• W Crispr: could in theory work but:

• The gene-editing would have to happen in stem cells
• It’s really hard to get the tools to the lungs
• Reparation relies on cells splitting and duplicating (?) and those in the lungs don’t usually do that

https://hihg.med.miami.edu/thromboticstorm/genetics-overview/single-gene-disorders

• https://ui.adsabs.harvard.edu/abs/2017APS..MARK29004B/abstract
• Background on the disease, how its treated right now
• We love a trusty overveiw:
• https://ui.adsabs.harvard.edu/abs/2017APS..MARK29004B/abstract

DESIGNING A CURE: an overview: also do not know where to put this but Cpf1 (the other version of Cas9) is waaaaay better (look at this)

David R. Liu Ted Talk: Can We Cure Genetic Diseases by Rewriting DNA

• Point Mutations and DNA Base Editing
• Point mutations account for a whole bunch of diseases, and we usually know where they are

• DEFINITION: single letter changes in our genome

• So, Crisper serves as molecular scissors, we can introduce new sequences of DNA around cut sites (I think that’s Homology Directed Repair, more on that later), according to this man, that sometimes ends up being hella faulty in terms of curing/ fixing a disease (does not work in most cells apparently, double check that)
• Process:

• So, he instead uses the “finding” aspect of CRISPR, and without cutting anything (just “unzipping” like the protein in transcription), uses “base editors” (two other engineered proteins attached to cas9) to just change one of the nucleotides and not the entire gene

• Those proteins are not naturally occurring

• This is done by rearranging the atoms from one DNA base to another
• They only change one strand, and then nick the other strand of DNA (the one w the not changed base) so the cell is tricked into replacing and fixing that strand as opposed to the edited one
• His lil cocktail of proteins can only change CG pairs into TA pairs tho

• They account for 14% of diseases caused by point mutations

• The change that would really benefit is switching AT to GC
• There’s been developments, they evolved their own protein (crazy story but irrelevant)

• Applications of this tool don’t stop at diseases, they also spread into agriculture
• The image below:

From Science Mag (pls check)

2/10/20:

2/24/20: Pick a disease to focus on and the bio and then use the CRISPR knowledge specifically to design a fix

• Focus on a certain disease

• Figure out what protein

• When its mutated:
• what it is normal:

• What is the mutation
• How would CRISPR help
• Start to think about the fix

3/2/20:

What is our fix? How do we accomplish it?

Sickle cell anemia https://ghr.nlm.nih.gov/condition/sickle-cell-disease (HbS disease

Hemoglobin S Disease, SCD, Sickle cell disorders, Sickling disorder due to hemoglobin S)

• A group of disorders that affect the hemoglobin (red blood cells that deliver oxygen to cells throughout the body)
• Red blood cells are shaped into sickle
• People who have sickle cell anemia have atypical hemoglobin molecules called hemoglobin S

• Distort red blood cells and sickle, or crescent, shape.

• Symptoms:

• Low number of red blood cells (anemia)

• When they sickle they break down prematurely
• Yellowing of the eyes and skin (signs of jaundice)
• Repeated infections

• Periodic episodes of pain

• Sickled blood cells are stiff and inflexible and get stuck in small blood vessels
• Deprive tissues and organs of oxygen -rich blood

• Organ damage

• Lungs
• Kidneys
• Spleet
• And brain

• High blood pressure in blood vessels that supply the lungs (pulmonary hypertension)

HOW IT HAPPENS:

• HBB gene
• Hemoglobin consists of four protein subunits

• Two subunits

• Alpha-globin (1 Alpha and 2 beta-globin)
• Beta-globin

• Result in different mutations in the HBB gene

• Hemoglobin S (HbS) — Most popular one
• Hemoglobin C (HbC)
• Hemoglobin E (HbE

• HBB gene mutations can result in low level beta-globin (beta thalassemia)
• People who have sickle cell disease, their beta globin subunits in hemoglobin is replaced with hemoglobin S
• In sickle cell anemia, which is a common form of sickle cell disease, hemoglobin S replaces both beta-globin subunits in hemoglobin.
• Abnormal versions of beta-globin can distort red blood cells into a sickle shape.

• The other beta-globin subunit is replaced with a different abnormal variant, such as hemoglobin C. For example, people with sickle-hemoglobin C (HbSC) disease have hemoglobin molecules with hemoglobin S and hemoglobin C instead of beta-globin. If mutations that produce hemoglobin S and beta thalassemia occur together, individuals have hemoglobin S-beta thalassemia (HbSBetaThal) disease.

INHERITANCE:

• It’s an autosomal recessive pattern which means both copies of the gene in each cell have mutations
• So the parents carry one copy of the mutated gene, but they typically do not show signs and symptoms of the condition.

Chromosome 11
https://rarediseases.org/rare-diseases/sickle-cell-disease/

• Non-conservative missense (meaning it has different properties than the other amino acid) -single point mutation
• Happens in the 6th amino acid in beta-globin is instead a valine (val) instead of a gluta (glu)
• Valine – hydrophobic
• Glutamic – hydrophilic
• Anemia

• Deficiency in RBCs
• Makes immature red blood cells and in creates new bone marrow

• Extramedualry hematopoiesis = blood red blood cells outside of the cell which can happen in the liver
• Vaso-occlusion = clogging capillaries
• Howell-Jolly Body
• Multi organ damage is the result

Cures/Treatment: https://www.npr.org/sections/health-shots/2019/11/19/780510277/gene-edited-supercells-make-progress-in-fight-against-sickle-cell-disease

• The edited cells are producing a crucial protein at levels that exceed what doctors thought would be needed to alleviate “the excruciating, life-threatening complications of the genetic blood disorder, the early data show”
• “the cells appear to have already started to spare the patient from the agonizing attacks of pain that are the hallmark of the disorder”
• They tried it in Germany and the CRISPR-treated patient normally needed more than 16 transfusions every year

• That patient too experienced health problems after the treatment but also recovered, and none is believed to have been caused by the treatment

• Fetal hemoglobin is a protein that is normally produced only by fetuses and newborn babies for a short time after birth. So scientists used CRISPR to edit a gene in bone marrow cells that had been removed. The edited cells were infused back into her system, and the editing change allowed the cells to start producing fetal hemoglobin again. The fetal hemoglobin will compensate for the genetic defect that has resulted in sickle cell disease and its abnormal form of adult hemoglobin.

• Starting at CRISPR at a young age can help

• But risks of that are probably higher

• blood infection, gallstones and abdominal pain after the grueling procedure, which involved the equivalent of a bone marrow transplant

https://www.pbs.org/newshour/science/first-crispr-treatment-for-sickle-cell-other-blood-disease-shows-early-benefits-in-two-patients

• The CRISPR/Vertex treatment, called CTX001, targets the two blood disorders in an indirect way.
• What they have done on previous patients is they’ve drawn blood and isolated hematopoietic (blood-producing) stem cells. Technicians ten zap the cells with a pulse of electricity (electroporation) opens a portal that the CRISPR complex passes through.
• The patient undergoes – busulfan treatment to destroy the mutation-carrying bone marrow cells remaining in the patient, the CRISPR’ed cells are infused into the bloodstream. If it makes it correct it makes a beeline for the marrow to set up production of healthy, hemoglobin-packed red blood cells