Pastor Lecture (CRISPR/Cas9)

Question 1

Before CRISPR, what are two double-stranded break repair mechanisms?

Answer 1

1) NHEJ (Non-homologous End Joining)
- Quick repair mechanism that rejoins broken DNA ends.
- Often introduces insertions or deletions (indels), leading to gene disruption.

2) HDR (homology Directed repair)
- Requires a DNA template to accurately repair the break, allowing precise editing.
- works best when cells are in the S Or G2 phase of the cell cycle.

Question 2

How could you target specific sites in the genome before CRISPR?

Answer 2

• Design a guide RNA (gRNA) that matches the sequence near the desired target site.
• It will bind to Cas9 and direct it to the specific DNA location, where Cas9 creates a double-stranded break.
• This break is repaired by NHEJ or HDR, depending on cell conditions and whether a repair template is provided.

Question 3

What are limitations of Classic Genomic Engineering? (pre-CRISPR)

Answer 3

• Limitations are that this method is dependent on sporadic genomic DNA breaks, these do not happen very often.
• these breaks are rare and unpredictable, making it difficult to target specific genes for editing.
• Requires selection markers and complex screening to identify cells with the desired genetic modification, this limitation set the stage for CRISPR.

Question 4

How was CRISPR discovered?

Answer 4

• Pathogen sequence
• First identified in bacterial genomes as a series of repeating DNA sequences, these were found to be part of a bacterial immune system against viruses (phages).
• Researchers notices between the repetitive sequences, there were spacer sequencers that matched DNA from viruses that had previously infected the bacteria. When the bacteria encountered the same virus again, it uses the CRISPR system to cut and destroy the viral DNA, preventing infection.

Question 5

Describe how the Natural CRISPR system works

Answer 5

• Tracr-RNA (trans-activating CRISPR RNA) and pre-crRNA (precursor CRISPR RNA) come together.
• Cas9 binds crRNA and tracrRNA, these are processed to mature form (forms complex).
• the crRNA guides the complex to the matching DNA sequence in the genome.
• Cas9 cutting: Cas9 recognizes a short DNA sequence next to the target site called PAM. Once bound to the target DNA, Cas9 makes a cut at the PAM site, creating a double-stranded break in the DNA.

Question 6

Describe the two-component CRISPR System

Answer 6

• Cas9 programmed by cRNA duplex:
  –> in the natural system, Cas9 is guided by a duplex formed by crRNA and tracrRNA to find and cut target DNA.
• Cas9 programmed by a single chimeric RNA, to simplify, scientist engineered a single guide RNA (sgRNA) by fusing crRNA and tracrRNA into one molecule. This single chimeric RNA guides Cas9 just as effectively, making the system easier and more efficient for gene editing.

Question 7

How would you demonstrate CRISPR in Mammalian Cells?

Answer 7

• Cells contain disrupted GFP sequence, CRISPR-HR fixes it and makes fluorescent cells.

Question 8

Efficient CRISPR HDR (homology directed repair) to edit Genomes.

Answer 8

• Adding donor DNA: When donor DNA (a repair template) is added alongside CRISPR, it enables HDR for precise genome editing.
• CRISPR-Streptavidin Fusion: Fusing streptavidin to Cas9 significantly increases HDR efficiency. It binds to biotin-labeled donor DNA, bringing it closer to the target site, enhancing the likelihood of accurate repair.

Question 9

dCas9

Answer 9

• Cas9 cuts stuff, but catalytically inactive “dead” Cas9 does not.
• you can conjugate other stuff to dCas9 (to have precise gene regulation/labeling without altering the DNA sequence itself)

Question 10

What are KRAB Zinc Fingers?

Answer 10

• Gene silencers
• Each KRAB protein in humans, target specific DNA binding sites.
• These proteins regulate transposon families and individual genes

Question 11

What is the mechanism of KRAB Zinc Finger Proteins?

Answer 11

• The KRAB domain of these proteins recruits KAP1 (KRAB-associated protein 1)
• KAP1 then attracts HDACs and H3K9 methyltransferases, leading to heterochromatin formation, tightly packed transcriptionally inacive chromatin state.
• especially inactive silencers because of their strong interaction with KAP1.

Question 12

CRISPRi

Answer 12

• silencing transcription via KRAB domain
• uses catalytically inactive dCas9 to bind to specific DNA sequences without cutting. dCas9 fuses to KRAB domain, recruits KAP1, attracts HDACs and H3K9 methyltransferases, forms heterochromatin, effectively silencing transcription at targeted gene site.

Question 13

CRISPRa

Answer 13

• VP16 is a powerful ‘trans-activator’
• Transcriptional activation mediated by tagging Cas9 with four copies of VP16 (VP64)

Question 14

The SunTag Tool

Answer 14

• will attract many molecules to one Cas9
• improve efficiency of CRISPRa

mechanism:
- Tagging: The target protein is fused with multiple SUnTag peptide repeats.

• Binding: SUnTag repeats recruit scFv antibodies linked to GFP or effector proteins.
• Signal Amplification: Multiple GFP or effector proteins bind to each SUnTag, amplifying the signal.
• Applications: Used for visualizing proteins or enhancing gene expression at target sites.

Question 15

How is CRISPR used in Medicine?

Answer 15

• Sickle cell anemia and B-thalassemia.
• mutations in B-globin gene, which leads to abnormal hemoglobin and red blood cell aggregation.
• CRISPR treatment strategy: extract bone marrow from the patient, use CRISPR to genetically repair the B-globin mutation, then reintroduce it to produce normal red blood cells. Transition from fetal to adult hemoglobin.
• BCL11A: transcription factor represses fetal hemoglobin (HbF) expression, transitioning the body to adult hemoglobin after birth. Restoring Fetal Hemoglobin (HbF) as a therapy.
• Strategy:
• mutations in the BCL11A enhancer reduce its expression, so there is more HbF. CRISPR can target the enhancer to lower BCL11A, boosting HbF and mitigating symptoms of sickle cell anemia and transfusion-dependent thalassemia.

Question 16

How is CRISPR Used in Drug Delivery?

Answer 16

• Hematopoietic system is easy to edit because the stem cells can be taken out to edit and reintroduced in the body. No other adult stem cells, such as muscle stem cells, can be edited in this manner. This creates a bioengineering challenge.
• –> Nanoparticle delivery for CRISPR for Duchenne Muscular Dystrophy:
• goal is to deliver Cas9 protein and sgRNA into cells in vivo to edit DMD gene.
• DMD (X-linked) genetic disorded caused by mutations in DMD gene on X chromosome.

Gold nanoparticles coated with thiol-DNA, Cas9, sgRNA, and donor DNA are taken into cells via endocytosis, with cationic lipids aiding the process. Once inside, the endosome collapses, releasing the gold particles, and cellular glutathione breaks the thiol-DNA link, freeing Cas9, sgRNA, and donor DNA to enter the nucleus for gene editing.

Question 17

How would you use SunTag System in CRISPRa?

Answer 17

In CRISPR, the SUnTag system is often combined with catalytically inactive Cas9 (dCas9) to amplify signals or enhance gene regulation. dCas9, guided to a target gene by an sgRNA, is fused with SUnTag peptides, which recruit multiple GFP or transcriptional activator proteins. This setup allows researchers to visualize the target gene or increase its expression, enabling live tracking or controlled gene activation without cutting the DNA.

Question 18

A patient has ß-thalassemia. Explain how you can you use CRISPR to treat this patient.

Answer 18

To treat β-thalassemia with CRISPR, hematopoietic stem cells are collected from the patient and genetically edited in the lab. CRISPR-Cas9 is used either to correct the mutation in the β-globin (HBB) gene or to knock out the BCL11A gene, which represses fetal hemoglobin (HbF). The edited cells, now capable of producing functional hemoglobin or compensating with HbF, are then transplanted back into the patient to improve red blood cell function and reduce symptoms.