CRISPR

week 7 and 8 at BioHack Academy Waag 2024

with Tom Peeters and Matthijs de Block

CRISPR, which stands for “Clustered Regularly Interspaced Short Palindromic Repeats,” is a technology that allows scientists to edit genes accurately and efficiently. It has revolutionized the field of genetics by enabling researchers to easily alter DNA sequences and modify gene function. The technology is based on a natural system used by bacteria as a form of immune defense against viruses. In the lab, CRISPR is used with a guide RNA and an enzyme called Cas9 (or other Cas proteins) to target specific sequences in the DNA, where it can cut the DNA strand and allow for genes to be added, removed, or altered.

CRISPR has a wide range of applications, including genetic research, the development of new treatments for genetic diseases, and improvements in agriculture to create crops with better yields, disease resistance, or climate resilience. Its ease of use and versatility make it a powerful tool in both basic research and in developing therapeutic solutions for complex diseases.

CRISPR cas-9

The bacterial gene lacZ is part of the lac operon in E. coli and other bacteria. This gene encodes the enzyme β-galactosidase, which is primarily involved in lactose metabolism. β-galactosidase catalyzes the hydrolysis of lactose into glucose and galactose, which the bacteria can then use as a source of energy.

The lacZ gene is often used in molecular biology as a reporter gene to monitor the expression of other genes. By linking lacZ to another gene of interest, researchers can easily assess the expression of the target gene by measuring the activity of β-galactosidase, which can be detected by its ability to cleave X-gal, a synthetic substrate, producing a blue color. This makes it a valuable tool for gene expression studies and genetic engineering.

Non-homologous end joining (NHEJ) is a pathway used by cells to repair double-strand breaks in DNA. This process is crucial for maintaining the stability of the genome, especially in the context of gene editing. Unlike homologous recombination, which uses a homologous sequence as a template for repair, NHEJ does not require a homologous template. Instead, it directly joins the broken DNA ends together.

In gene editing, NHEJ is often exploited to introduce mutations or deletions at specific genomic locations. When a gene-editing tool like CRISPR-Cas9 creates a double-strand break in DNA, the cell may use NHEJ to repair the break. Because NHEJ can rejoin the DNA ends in a somewhat error-prone manner, small insertions or deletions (indels) can occur at the repair site. These indels can disrupt the gene’s function, effectively “knocking out” the gene. This makes NHEJ a powerful tool for researchers aiming to study gene function or develop genetic therapies by selectively disabling genes.

Introducing a mutation into E. coli that involves X-gal is typically part of a process used in molecular biology to identify and differentiate bacterial colonies based on their ability to metabolize the substrate X-gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside).

EXPERIMENT: we have an E. coli strain that contains several genetic components:

1. Functional lacZ Gene: This gene encodes the enzyme β-galactosidase, which breaks down X-gal to produce a blue color in colonies.
2. Plasmid with HDR Gene: This plasmid carries a gene that enables Homology-Directed Repair (HDR), a method used in genetic engineering to introduce specific changes to the DNA sequence. This gene is controlled by an arabinose-inducible promoter, meaning its expression can be triggered by the presence of arabinose in the medium.
3. Antibiotic Resistance Gene: Both plasmids also carry genes that confer resistance to a specific antibiotic, which allows for the selection of cells that have successfully taken up the plasmid.

Experimental Consideration

To experimentally introduce these mutations, techniques such as CRISPR-Cas9 or other forms of site-directed mutagenesis might be used. The HDR system in the plasmid can facilitate precise editing by providing a template for repair after DNA cleavage by CRISPR or another nuclease.

Observational Outcomes

• Blue Colonies: Indicate that the lacZ gene is still functional and expressing β-galactosidase that can metabolize X-gal.
• White Colonies: Suggest that the lacZ gene has been disrupted by the mutation, preventing the production of functional β-galactosidase.

This setup allows you to study the effects of genetic mutations on protein function in a controlled manner, and it provides a visual way to assess the impact of different types of genetic alterations on enzyme activity. The use of X-gal as a reporter makes it easy to identify and categorize the mutations based on their phenotypic expression in the bacteria.

step by step

1. X-gal: X-gal is a colorless substrate that, when cleaved by the enzyme β-galactosidase, produces a blue product. This enzyme is encoded by the lacZ gene in the lac operon of E. coli.
2. Mutation and Blue Color Formation:
   • If E. coli is genetically engineered or naturally capable of expressing the lacZ gene, it will produce β-galactosidase.
   • When these bacteria are grown on a medium containing X-gal, the β-galactosidase enzyme breaks down the X-gal, resulting in the release of a blue dye. This turns the colonies blue.
   • This process is often used in cloning experiments to distinguish between bacteria that have successfully incorporated foreign DNA (which disrupts the lacZ gene, leading to white colonies because they can’t metabolize X-gal) and those that have not (which remain capable of metabolizing X-gal and turn blue).
3. Biotechnological Applications:
   • This technique is particularly useful in genetic engineering for blue/white screening, where plasmids (vectors for carrying foreign genetic material) are often constructed with an interrupted lacZ gene. Only bacteria that take up plasmids without the foreign insert will have a functioning lacZ gene and will turn blue on X-gal plates.

Thus, the blue color that appears when introducing a mutation into E. coli with X-gal indicates that the bacteria are expressing the lacZ gene and successfully metabolizing X-gal, which is an indicator of their genetic makeup regarding the lac operon.

dna -rna -protein

There are several types of genetic mutations that can affect DNA, RNA, and ultimately protein function. Understanding these mutations is crucial in genetics because they can lead to changes in biological functions and phenotypes. Here are the main types of mutations:

1. Point Mutation (Substitution)
   • Silent Mutation: Alters a DNA base but does not change the amino acid sequence due to the redundancy of the genetic code.
   • Missense Mutation: Changes a single DNA base pair, which results in the substitution of one amino acid for another in the protein. This can affect the protein’s function, depending on the importance of the altered amino acid.
   • Nonsense Mutation: A substitution that changes a codon for an amino acid into a stop codon, leading to premature termination of protein synthesis. This usually results in a nonfunctional protein.
2. Insertions and Deletions
   • Frameshift Mutation: Insertion or deletion of a number of nucleotides not divisible by three, which alters the reading frame of the gene. This can dramatically change the amino acid sequence from the mutation site forward, likely resulting in a nonfunctional protein.
   • In-frame Insertions and Deletions: Insert or remove nucleotides in multiples of three. These mutations do not alter the reading frame but add or remove amino acids in the protein.
3. Duplication
   • Parts of the DNA molecule are duplicated, resulting in multiple copies of a segment of DNA. This can lead to gene dosage effects or create new gene functions.
4. Expansions
   • Trinucleotide Repeat Expansions: The number of repeats of three nucleotides (e.g., CAG, CTG) increases in the gene. Diseases like Huntington’s disease and myotonic dystrophy are caused by such expansions, affecting gene function through various mechanisms.
5. Transversions and Transitions
   • Transversion: A purine (A, G) is substituted by a pyrimidine (C, T), or vice versa.
   • Transition: A purine replaces another purine, or a pyrimidine replaces another pyrimidine. Transitions are more common than transversions and can have varied effects on the protein, depending on the context of the mutation.

Each type of mutation has a different potential impact on the gene product (protein) and its function, which can range from benign to causing serious genetic disorders or contributing to the development of cancer.

II. Art and Immunology with MARTHA DEMENEZ

Methodology reflexion:

Design thinking is a problem-solving approach that emphasizes human-centered, iterative, and practical methods to develop innovative solutions. It consists of five main phases:

1. Empathize: The first step is about understanding the needs, experiences, and challenges of the users for whom you are designing. This involves engaging with users, observing their behaviors, and developing empathy through interviews, surveys, and observation. This phase is crucial for gathering insights about the user’s experiences and needs.
2. Define: In this phase, you synthesize the information gathered during the Empathize phase to define the core problems you have identified. This is typically articulated as a point-of-view (POV) problem statement that focuses on the user, their needs, and the insights and challenges that are identified. It’s about bringing clarity and focus to the design space.
3. Ideate: After defining the problem, you brainstorm a range of creative solutions without constraints. This phase encourages thinking outside the box and generating a broad set of ideas to explore and select from. Techniques like brainstorming, SCAMPER, and mind mapping can be particularly useful here to expand the solution space.
4. Prototype: This stage involves turning ideas into tangible products. Prototypes can be scaled-down versions or specific aspects of the product to test particular features. The goal here is not to develop a fully functioning model, but rather to create a representation of the solution that can be used to test its viability.
5. Test: The final phase involves testing the prototype with real users to understand its efficacy and gather feedback. Testing is iterative, meaning that insights gained will often lead you back to previous stages. Based on the feedback, the prototype may be refined, alterations may be made, and further tests might be conducted to improve the solution.

Each stage of the design thinking process is iterative, allowing you to continually refine your approach and your understanding of the problem and users, leading to more refined solutions. This method ensures that the solutions are as human-centered, practical, and innovative as possible.

key questions:

“learn from the exhibition how to devise better triggers, create an umbrella team-identity, political or personal level: who are you, who you want to become? how we relate to other species, how we build the understanding of our own identity “

“Genetic manipulation: My identity is not based solely on my genealogical tree. Identity is immunology—cell lines.

What becomes immortal stops being human.

The immune system distinguishes between self and non-self.

I don’t want to lose myself to another person.”

III. BESTIARY

with Maro Pebo

A bestiary is a collection or compendium of descriptions and illustrations of real and mythical animals, often with accompanying moral lessons or allegories. Originating in the Middle Ages, bestiaries were popular manuscripts that combined natural history, folklore, and moral instruction, usually organized in an illustrated book.

If a bestiary were created using CRISPR-Cas9, it could be imagined in several ways:

1. Modern Bestiary of Real Creatures: This version would describe and illustrate animals that have been genetically edited using CRISPR-Cas9. It might detail the modifications made to improve certain traits, such as disease resistance, size, or productivity. Each entry could include:
   • Name of the animal
   • Description of genetic modifications
   • Purpose of the modifications
   • Illustrations showing the changes
   • Potential ethical considerations
2. Futuristic Bestiary of New Species: This version would include entirely new species created through CRISPR-Cas9 technology. These could be speculative or fictional creatures designed for various purposes, such as environmental restoration, agriculture, or even companionship. Each entry might feature:
   • Name and classification of the new species
   • Detailed description of its physical characteristics
   • Genetic blueprint and CRISPR modifications used
   • Illustrations of the new species in its environment
   • Potential uses and ethical implications
3. Mythical Bestiary Reimagined with CRISPR: This version could reimagine mythical creatures like dragons, unicorns, or griffins as if they were created using modern genetic engineering techniques. Each entry could include:
   • Name and mythological background
   • Description of how CRISPR could theoretically create such a creature
   • Scientific explanation of the genetic modifications required
   • Illustrations blending traditional mythology with modern genetics
   • Discussion of the feasibility and implications of creating such creatures

An example entry for a modern CRISPR bestiary might look like this:

Name: Glow-in-the-Dark Rabbit

Description: The glow-in-the-dark rabbit is a genetically modified rabbit that exhibits bioluminescence. Using CRISPR-Cas9, scientists introduced genes from jellyfish into the rabbit’s DNA, causing it to glow green under UV light.

Purpose of Modifications:

• Research: To study gene expression and the effects of genetic modifications.
• Public Engagement: To raise awareness about genetic engineering.

Illustrations:

• Diagram showing the insertion of jellyfish genes into rabbit DNA.
• Illustration of the rabbit glowing in the dark.

Ethical Considerations:

• Animal welfare concerns regarding the modification.
• Potential environmental impact if such animals were released into the wild.

This blend of scientific detail, illustrative content, and ethical discussion would make for a comprehensive and fascinating bestiary for the modern age.