Case 9 - BBS2042

Case 9 - CRISPR

1. Learning Goals

  • CRISPR: What it stands for and its function in bacteria.

  • History of CRISPR.

  • CRISPR-Cas9: Components and structure.

  • CRISPR Mechanism in Bacteria: Discuss three types, focus on type 2.

  • Gene Editing: Function of CRISPR in gene editing.

  • Second-Generation CRISPR Technologies.

  • Limitations of CRISPR Technology.

  • BRCA1/2 Function: Overview of mutations leading to cancer (limited detail).

  • CRISPR Applications: Potential for curing breast cancer.

2. What is CRISPR?

2.1 What CRISPR Stands For

  • CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats.

    • Definition: Describes a specific structure of DNA in bacterial genomes.

    • Details:

    • Clustered: Sequences are grouped together in one region.

    • Regularly interspaced: Repeated sequences interspersed with unique spacer sequences.

    • Short palindromic repeats: DNA sequences that are similar when read forwards and backwards.

  • Typical CRISPR Locus Includes:

    1. Repeats: Identical palindromic DNA sequences.

    2. Spacers: Unique sequences derived from prior viral infections.

    3. Cas Genes: Genes encoding CRISPR-associated proteins (e.g., Cas9).

2.2 Natural Function of CRISPR in Bacteria

  • Functions as an adaptive immune system against:

    • Bacteriophages: Viruses that infect bacteria.

    • Plasmids: Extrachromosomal DNA elements.

  • Mechanism: Allows recognition and destruction of viral DNA upon reinfection.

3. History of CRISPR

3.1 First Observation of CRISPR Sequences (1987)

  • Discoverer: Yoshizumi Ishino and colleagues.

  • What was Found:

    • A peculiar DNA region with short repeated sequences separated by unique spacers.

  • Context: Discovery was accidental; the function was unknown.

3.2 Discovery in Many Microbes (1990s)

  • Key Scientist: Francisco Mojica.

  • Observations:

    • Similar patterns found in diverse bacteria and archaea.

    • Structure conservation observed.

  • 2002: The official term “CRISPR” proposed.

  • Identification of nearby CRISPR-associated genes (Cas genes).

3.3 CRISPR Identified as a Bacterial Immune System (2005–2007)

  • Discovery of viral DNA matching spacer sequences suggested:

    • Bacteria retain fragments of viral genomes as genetic memory.

  • Experimental Proof (2007): Research by Philippe Horvath demonstrated that:

    • Adding viral DNA spacers enabled bacteria to resist those viruses, confirming CRISPR as an immune system.

3.4 Discovery of the CRISPR-Cas9 Mechanism (2011–2012)

  • Key Scientists: Jennifer Doudna and Emmanuelle Charpentier.

  • Key Discoveries:

    1. Cas9 can be directed by a guide RNA.

    2. RNA can target any DNA sequence.

    3. Cas9 executes a DNA cut at that location.

  • Publication: Work released in Science, marking CRISPR as a programmable gene-editing tool.

3.5 First Genome Editing in Human Cells (2013)

  • Contributors: Feng Zhang and George Church.

  • Significance: Demonstrated CRISPR-Cas9 editing in human cells.

  • Applications Expanded:

    • Gene knockout.

    • Gene insertion.

    • Disease modeling.

3.6 Explosion of Research and Biotechnology (2013–2019)

  • CRISPR rapidly became the dominant gene-editing tool.

  • Developments included:

    • Improved Cas enzymes.

    • Base editing.

    • Prime editing.

    • CRISPR diagnostics.

  • CRISPR applications in:

    • Studying gene function.

    • Creating animal disease models.

    • Agricultural engineering.

    • Drug discovery.

3.7 First Therapeutic Applications in Humans (2016–2018)

  • Early clinical trials based on:

    • Cancer immunotherapy.

    • Blood disorders.

    • Inherited diseases.

  • Notable Success: Treatment of Sickle Cell Disease.

    • Edited hematopoietic stem cells restoring fetal hemoglobin production.

3.8 First CRISPR-Based Medicine Approval (2023–2024)

  • Milestone Event: Approval of first CRISPR therapy: Casgevy.

  • Developers: Vertex Pharmaceuticals and CRISPR Therapeutics.

  • Conditions Treated:

    • Sickle Cell Disease.

    • Beta Thalassemia.

  • Treatment Approach:

    1. Remove patient stem cells.

    2. CRISPR edits the BCL11A gene.

    3. Return edited cells to the patient.

    4. Restoration of fetal hemoglobin production and symptom reduction.

3.9 Recognition with the Nobel Prize (2020)

  • Awardees: Jennifer Doudna and Emmanuelle Charpentier.

  • Achievement: Recognized for the development of CRISPR-Cas9 genome editing.

3.10 Quick Timeline Summary

  • Discovery Events:

    • 1987: Repeated DNA sequences discovered.

    • 1990s: CRISPR found across microbes.

    • 2002: Term "CRISPR" introduced.

    • 2005: The viral origin of spacers discovered.

    • 2007: Experimental proof of immune function.

    • 2012: Programmable CRISPR-Cas9 editing developed.

    • 2013: Editing demonstrated in human cells.

    • 2016–2018: First human clinical trials.

    • 2023–2024: First approved CRISPR therapy.

4. Structure of CRISPR-Cas9

4.1 Main Components of the CRISPR–Cas9 System

  • Essential Components:

    1. Cas9 protein.

    2. Guide RNA (gRNA).

    3. Target DNA containing a PAM sequence.

  • Functionality: Together, these enable sequence-specific DNA cleavage.

4.2 Cas9 Protein (the Molecular Scissors)

  • Type: DNA endonuclease capable of cutting double-stranded DNA.

  • Common Variant: SpCas9 from Streptococcus pyogenes.

  • Structure: Comprises two major functional lobes:

    1. Recognition (REC) Lobe:

    • Binds guide RNA.

    • Positions target DNA.

    1. Nuclease (NUC) Lobe:

    • Contains domains responsible for DNA cleavage.

      • HNH Domain: Cuts the DNA strand complementary to the guide RNA.

      • RuvC Domain: Cuts the non-complementary DNA strand.

  • Outcome: Production of a double-strand break (DSB).

  • PAM-Interacting Domain: Recognizes PAM sequence on DNA.

4.3 Guide RNA (gRNA)

  • Function: Determines the site of Cas9 cuts.

  • Components:

    1. crRNA (CRISPR RNA): Contains sequence complementary to viral DNA.

    2. tracrRNA (trans-activating CRISPR RNA): Stabilizes the Cas9 complex.

  • In Genome Editing: Both are fused into a single molecule known as a single guide RNA (sgRNA).

4.4 Target DNA

  • Definition: The DNA sequence designated for editing.

  • Requirements for Cas9 to Cut:

    • Must have a sequence complementary to the 20 nt guide RNA.

    • Must contain a PAM sequence next to the target DNA.

  • For SpCas9: PAM is defined as (5'-NGG-3'), where:

    • N: Any nucleotide.

    • GG: Two guanines.

4.5 Why the PAM Sequence is Important

  • Two Main Functions:

    1. Prevention of self-targeting: Cas9 does not cut the bacterium's own genome.

    2. Initiation of DNA binding: Cas9 first scans for PAM motifs before binding to DNA.

5. Mechanisms of CRISPR-Cas

  • Overview: CRISPR–Cas systems act as adaptive immune systems protecting bacteria and archaea from viral infection by recognizing and destroying invading nucleic acids.

  • Process Steps: 1. Adaptation (insertion of foreign DNA into the CRISPR array).

  1. Biogenesis (maturation of crRNA).

  2. Interference (destruction of invading DNA).

5.1 Type I CRISPR System — Cascade + Cas3

  • Components:

    • Cascade Complex: Multi-protein complex binding crRNA and searching for targets.

    • Cas3: Helicase-nuclease enzyme responsible for DNA degradation.

  • Recognition Requirement: Involves PAM sequences.

5.2 Type II CRISPR System — Cas9

  • Key Components:

    • CRISPR array: Comprises genomic repeats and spacers.

    • Cas9 protein: Acts as RNA-guided endonuclease.

    • tracrRNA: Required for crRNA maturation and Cas9 targeting.

    • RNase III: Host enzyme involved in RNA processing.

  • Process: Includes steps from adaptation to target search and interference.

Step 1: Adaptation (Spacer Acquisition)

  • Capture of foreign DNA (phage DNA) fragments for new spacers.

Step 2: Expression of the CRISPR Locus

  • Transcription into pre-crRNA.

Step 3: crRNA Maturation

  • Involves tracrRNA hybridization with pre-crRNA, leading to the formation of individual crRNA–tracrRNA duplexes by RNase III cleavage.

Step 4: Complex Assembly

  • Formation of the Cas9–guide RNA complex.

Step 5: Target DNA Search

  • Cas9 scans for PAM, binding for DNA unwinding.

Step 6: R-loop Formation

  • Formation of an RNA–DNA hybrid occurring once complementary binding is confirmed.

Step 7: Structural Activation of Cas9

  • Confirmation leads to activation of nuclease domains and cleavage.

Step 8: DNA Cleavage

  • Two cuts via HNH and RuvC domains, producing a DSB.

Step 9: After Cleavage

  • Cleavage leads to degradation of viral genome, preventing viral replication.

5.3 Type III CRISPR System — Csm/Cmr + Cas10

  • Unique Capability: Targets both RNA and DNA.

  • Components:

    • crRNA

    • Csm or Cmr multiprotein complex

    • Accessory nucleases

    • Cas10

  • PAM Requirement: Not necessary in Type III systems.

6. Gene Editing with CRISPR-Cas9

  • Major Applications:

    1. Functional Genomics: Gene knockout studies.

    2. Disease Modeling: Create models for human diseases.

    3. Gene Therapy: Correct mutations linked to diseases.

    4. Agriculture: Develop disease-resistant and drought-resistant crops, and improve livestock traits.

6.1 Designing the Guide RNA

  • Selection Process: Identify a 20 nucleotide sequence adjacent to a PAM.

  • sgRNA Structure: Comprises a guide sequence and scaffold region for Cas9 binding.

6.2 Delivery Into the Cell

  • Components: Delivery of Cas9, gRNA, and optionally repair template.

  • Methods: Plasmids, viral vectors, lipid nanoparticles, or ribonucleoprotein complexes.

6.3 Target Recognition

  • Process:

    1. Cas9 binds PAM.

    2. Locally unwinds DNA.

    3. Guide RNA checks for complementarity.

6.4 DNA Cleavage

  • Confirmation activates cleaving domains leading to DSB formation.

6.5 DNA Repair Mechanisms

  • Repair Pathways:

    • Non-Homologous End Joining (NHEJ):

    • Mechanism involves direct rejoining of broken ends and often results in indels.

    • Homology-Directed Repair (HDR):

    • Utilizes a repair template for precise edits.

6.6 Cellular Outcome

  • Post-repair, the modified genome continues through cell division with edits inherited by daughter cells.

7. Second Generation CRISPR Technologies

  • Overview: Engineered versions of Cas9 created to enhance precision and reduce repair limitations.

7.1 Dead Cas9 (dCas9): CRISPR for Gene Regulation

  • Modification: Mutations disable Cas9 cutting.

  • Applications:

    • CRISPR interference (CRISPRi): Gene repression.

    • CRISPR activation (CRISPRa): Gene activation via fusing activators to dCas9.

7.2 Base Editing

  • Mechanism: Allows for single nucleotide edits without creating DSBs.

  • Types:

    • Cytosine Base Editors (CBE): Convert C to T.

    • Adenine Base Editors (ABE): Convert A to G.

7.3 Prime Editing

  • Capabilities: Can perform substitutions, insertions, deletions without inducing DSBs.

7.4 CRISPR Epigenome Editing

  • Mechanism: Modifies epigenetic marks instead of DNA sequences to influence gene expression.

8. Limitations of CRISPR Gene Editing

8.1 Off-Target Effects

  • Concern: Potential unwanted cuts leading to mutations or loss of gene function.

8.2 Delivery Challenges

  • Delivery efficiency varies, particularly concerning specific tissues, immune responses, and avoiding unintended cells.

8.3 Low Efficiency of Precise Editing (HDR)

  • HDR is less frequent than NHEJ, making precise edits more challenging.

8.4 Mosaicism

  • Results from editing after cell division, leading to incomplete therapeutic effects.

8.5 Large Genomic Alterations

  • DSBs may lead to unintended large-scale variations such as deletions or rearrangements.

8.6 Immune Responses

  • Recognition of bacterial Cas proteins as foreign leads to potential rejection or inflammation.

8.7 Tumor Heterogeneity in Cancer

  • Variability in cancer mutations complicates targeted CRISPR treatments.

8.8 Limited Targeting Range (PAM Requirement)

  • PAM sequence dependencies limit potential target sites.

9. BRCA1 and BRCA2

  • Overview: Tumor suppressor genes vital for DNA stability and DSB repair through homologous recombination.

9.1 How BRCA Mutations Lead to Cancer

  • Mechanism of Action: Impairment of accurate repair increases risk of mutations leading to genomic instability.

  • Cancer Risks:

    • Breast cancer: ~60-80%

    • Ovarian cancer: ~20-40%

9.2 Can CRISPR Cure Breast Cancer?

  • Potential: Exploration of correcting BRCA mutations with CRISPR, challenges include delivery, off-target effects, and gene variability.

10. Ethics of CRISPR

10.1 Germline vs. Somatic Editing

  • Somatic Editing: Generally accepted for severe diseases.

  • Germline Editing: Controversial due to potential for altering future generations.

10.2 Designer Babies & Enhancement

  • Concerns: Risks of non-therapeutic enhancements with gene editing; implies potential consumer eugenics.

10.3 Safety & Off-Target Effects

  • Technical limitations may lead to severe health issues if off-target mutations are triggered.

10.4 Inequality & Access

  • High costs may limit CRISPR access to the wealthy, creating disparities.

10.5 Environmental & Biosecurity Risks

  • Gene editing in agriculture could have unpredictable effects in ecosystems.

10.6 "Playing God" & Genetic Integrity

  • Ethical dilemmas arise from manipulating human genes and natural existence.

10.7 Proposed Solutions & Ethical Frameworks

  • International Regulation: Calls for strict guidelines to prevent rogue applications.

  • Public Dialogue: Societal engagement to determine acceptable gene editing uses.

  • Focus on Therapy: Encourage serious medical research while pausing enhancements for reproductive purposes.