BIOL3060 Lecture #19 CRISPR-Cas9

Staggering Impact

• Didn't invent CRISPR, discovered it

• Life existed on Earth 4 billion years

• 99.9% of time past before homo sapiens arrived

• Then didn’t know anything about DNA until 65 years ago (only 1 lifetime)

  • Learned structure of DNA, genetic info copying and storage, how to read/write DNA, and now learned how to edit DNA

Evolution of CRISPR-Cas9

• Didn’t invent CRISPR, discovered it in bacteria 

• 1987: Repetitive DNA sequences found in E. coli

• Name

  • Clustered

  • Regularly

  • Interspaced

  • Short

  • Palindromic

  • Repeats

• 2005: Mojica proposed that CRISPR sequences serves as a bacterial immune system

• 2012: Doudna and Charpentier publish a paper describing how you can progran CRISPR to edit specific genes

  • Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity

How Does CRISPR Work in Bacteria?

• Bacteriophages inject genetic material into the cell, hijack cell(transcription/translation machinery)

• Bacteria has to find a way to defend against viral DNA

• Cas1 and Cas2 recognize viral DNA and cut it into short sequences

  • Viral sequences are inserted in between repeated palindromic sequences: Spacer acquisition (create molecular memory)

  • CRISPR RNA Biogenesis: cRNA is CRISPR RNA and trans-activating RNA, both created and come together to make structure wit- h hairpin loop: able to recognize viral sequence and bnd to CAS9 (a nonspecific DNA nuclease) 

    • Hairpin loop structure is a surveillance complex, which moves around cell looking for the viral sequence which is recgnizes with complementary base pairing and then nuclease Cas9 can double strand break viral genome

  • Interference: process by which surveillance complex with Cas9 cuts DNA

• Found that you can cut whatever you want, just have to provide it with the 20 nucleotide guide sequence

• Tracker RNA can be attached to CRISPR RNA, so can provide a single guide RNA

• Cas9 protein creates double stranded breaks in DNA

  • After break is made, goal is to repair it right away

From Bacterial Immune System to Precise Molecular Scissors

• Target the right gene

• Bind the target gene

• Cut the DNA with complexed Cas9 protein

• Repair and edit the sequence

Two Mechanisms for Repairing Double-Stranded Breaks

• A: Non-homologouse end joining

  • Quick but error-prone

  • Can cause small insertions or deletions that disrupt gene function

  • Used to disable genes

    • Used for knockouts: helps people figure out what the gene does bc can see what happens when it’s not working

• B: Homology Directed Repair

  • Precise but slow

  • DNA template required

  • A custom DNA template can be inserted

  • Used for corrections, replacements, or insertions 

CRISPR-Cas9 Components

• Key Components

  • Gene for Cas9

    • Complexed with Nuclear localization signal: so Cas9 will go to the nucleus, where the DNA is

  • 20 nt target RNA sequence

  • Selection marker: makes the cell fluorescent green so you know CRISPR made it in to the cell

CRISPR-Cas9 Delivery Into Cells (In vitro: in a dish)

• CRISPR-Cas9 on a plasmid

• Ways to get plasmid into the cell

  • Microinjection: Take a needle and inject into cell

  • Electroporation: Electrocute the cell which creates transient holes in the cell for the plasmid to slip in 

    • Could potentially kill the cell, only 10% efficiency

  • Viral Mediated Delivery Vehicles

    • Hallow out the inside of specific vral vectors, put plasmid in, then infect the cell with the virus

  • Non-Viral Mediated Delivery Vehicles

    • Cover DNA with lipid nanoparticles (LNPs) that will fuse to the cell membrane and allow entry into the cell

The In vivo (in a person) Delivery Problem

• The gene therapy delivery problem in humans is that it is VERY difficult to efficiently deliver these therapeutic genes directly into a human (need to get it to many cells, not just one)

  • If, for example, trying to address something in lungs like cystic fibrosis, can use a virus that affects the respiratory tract only, like using a flu or common cold virus (use of viral mediated delivery vehicles)

  • Can also use the LNPs lipid nanoparticles

Genome Editing with CRISPR-Cas9

• Sickle Cell Anemia is caused by a single base change in a gene (approved by FDA)

  • We have access to bone marrow, can get CRISPR into cells and return bone marrow

  • Collect blood stem cells

  • Modify cells

  • Inject back to body through infusion

  • Price: 2.2 or 3.1 million dollars for a single infusion

• CRISPR Assisted CAR-T Cell Therapy (for cancer)

  • Can take T-cells out in blood and provde them with CAR-T gene that recognizes cancer cells so then Cas9 can recognize cancer cells and kill them

  • Price: 500k for infusion, but more like 1M with indirect cost of hospitalization

How Can CRISPR Be Used?

• Edits in somatic cells stop with you, so any change to disease in you will not be passed to children, so children can still get the disease

  • IMPORTANT: differences in editing babies v. embryos, somatic v. germ line

Debates about CRISPR

• Concerns:

  • Access issues: only the wealthy will be able to cure their diseases, widens healthcare disparity

  • Can’t ensure there won’t be some off-site mutations, potential detrimental longterm affects

  • Religious reasons

  • Debate on what is or isn’t a “serious disease” that needs eradicated

  • Ethics of using CRISPR for enhancement (like attractiveness and intelligence) → Crosses into Eugenics

  • Crossing into the germline has severe ethical complications

    • Chinese scientist did already genetically engineer babies

• Benefits:

  • Therapeutic applications: can be used to eliminate disease from the human genome