Advanced Yeast Techniques

CRISPR • Fluorescent Dyes • Lifespan Assays • Aging Research Tools

Yeast (Saccharomyces cerevisiae) is one of the most powerful organisms for genetic and aging research.
It is easy to grow, safe, genetically tractable, and shares many conserved pathways with humans.

This page introduces advanced techniques used in modern yeast biology:

• CRISPR gene editing
• fluorescent dyes and microscopy
• replicative & chronological lifespan assays
• mitochondrial and vacuole staining
• stress assays
• selection markers

Most of these techniques require more equipment than a beginner home lab, but they are essential to understand yeast aging and research methods.

1. CRISPR Gene Editing in Yeast

Yeast was one of the first organisms where CRISPR worked extremely well.
It is widely used for:

• deleting genes
• adding genes
• tagging proteins
• modifying promoters
• introducing point mutations

1.1. Why CRISPR is easy in yeast

• Yeast naturally integrates DNA via homologous recombination
• Short homology arms (~40–60 bp) are enough
• Plasmids are easy to transform
• Yeast grows fast and forms colonies quickly
• Selection markers are abundant

1.2. Basic components

A typical CRISPR edit requires:

• Cas9 (nuclease)
• gRNA targeting a specific gene
• Repair template with desired sequence
• Selectable marker (URA3, LEU2, HIS3, KanMX, etc.)

1.3. What you can edit

• Delete a gene completely
• Replace a promoter
• Insert GFP (or other fluorescent tags)
• Create temperature-sensitive alleles
• Modify metabolic pathways
• Change regulators of aging (SIR2, TOR, SCH9)

1.4. Optical visibility

CRISPR itself is invisible.
But edits may create visible phenotypes like:

• slow growth
• increased budding defects
• altered vacuole morphology
• fluorescence if tagged with GFP

2. Fluorescent Dyes and Stains

Fluorescence microscopy allows visualization of structures too small for conventional optical microscopes.

2.1. Common fluorescent dyes in yeast

DAPI

• Stains DNA (nucleus)
• Emits blue fluorescence
• Requires a fluorescence microscope
• Toxic → requires proper safety

MitoTracker

• Labels mitochondria
• Shows mitochondrial fragmentation (aging phenotype)

FM4-64

• Stains vacuolar membranes
• Used to study endocytosis
• Beautiful red fluorescence

Rhodamine 123

• Mitochondrial membrane potential dye
• Indicates mitochondrial health

Calcofluor White

• Binds yeast cell wall chitin
• Highlights bud scars
• Good for counting replicative lifespan events

2.2. What you can see with fluorescent dyes

• mitochondria fragmentation
• vacuole enlargement
• nuclear migration
• bud scars
• membrane dynamics

2.3. Requirements

• fluorescence microscope
• filters matching excitation/emission wavelengths
• proper handling of dyes

Not possible with a simple optical microscope.

3. Replicative Lifespan Assays (RLS)

Replicative lifespan = how many daughters a mother yeast cell can produce before senescence.

Typical lifespan: - 20–30 divisions for S. cerevisiae

3.1. How RLS assays are done

1. Isolate a single mother cell
2. Observe budding
3. Remove each daughter manually (micromanipulator)
4. Count total buds before the mother stops dividing

3.2. Aging hallmarks visible in RLS

• mother cell enlargement
• slower budding
• increased vacuole size
• mitochondrial dysfunction
• accumulation of bud scars

3.3. Why RLS is important

RLS models aging in dividing human cells:

• stem cells
• epithelial cells
• germline lineage

4. Chronological Lifespan Assays (CLS)

Chronological lifespan = how long non-dividing yeast survive in stationary phase.

Models aging in non-dividing human cells:

• neurons
• muscle cells
• heart cells

4.1. How CLS is measured

1. Grow yeast culture
2. Let it reach stationary phase
3. Over days/weeks, take samples
4. Plate cells to test viability
5. Count colonies (CFUs)

4.2. What CLS measures

• stress resistance
• metabolic resilience
• mitochondrial health
• ROS management

5. Mitochondrial Assays

Mitochondria are central to yeast aging.

Techniques include:

5.1. MitoTracker or Rhodamine dyes

Reveal:

• fragmentation
• network collapse
• membrane potential loss

5.2. Oxygen consumption assays

Measure mitochondrial respiration:

• Seahorse analyzer (advanced)
• Chemical dyes for redox states

5.3. mtDNA manipulation

Deleting or mutating mitochondrial genes yields:

• “petite mutants” (non-respiring)
• slow-growing but long-lived or short-lived phenotypes

6. Vacuole Staining & Aging

Yeast vacuoles change dramatically during aging.

Techniques:

• FM4-64 for membrane
• CMAC for vacuole lumen
• pH-sensitive dyes for acidification

Aging vacuoles show:

• increased size
• altered acidity
• slower fusion dynamics

These correlate strongly with lifespan.

7. Stress and Damage Assays

Yeast are ideal for studying cellular stress processes:

• oxidative stress
• heat shock
• osmotic shock
• DNA damage
• nutrient starvation
• ER stress

7.1. Reactive Oxygen Species (ROS) dyes

Examples:

• DHE
• H2DCFDA

Reveal oxidative stress during aging.

7.2. Heat shock protein induction

Measure expression of:

• Hsp70
• Hsp104

Indicates proteostasis stress.

8. Yeast Transformation Techniques

Introducing DNA into yeast can be done by:

8.1. Lithium acetate transformation (standard)

Simple and widely used.

8.2. Electroporation

More efficient but needs specialized equipment.

8.3. Spheroplast transformation

Yeast cell wall removed → DNA introduced → wall regenerated.
Used for difficult transformations.

9. Selection Markers and Reporters

Common selectable markers:

• URA3
• LEU2
• HIS3
• TRP1
• KanMX (G418 resistance)

Common reporters:

• GFP
• YFP
• RFP
• Luciferase
• LacZ

Used to track expression, localization, and pathway activation.

10. What You Can See Under a Light Microscope

Regular optical microscopes cannot see:

• CRISPR edits
• DNA
• proteins
• mitochondria
• organelles (except vacuole sometimes)
• fluorescent signals (requires filters)

But can see:

• budding
• large vacuoles
• mother/daughter size differences
• stress-induced shape changes
• aging phenotypes (limited)

To see advanced features, fluorescence microscopy is required.

11. Quick Beginner Summary

• Yeast are extremely powerful for genetic and aging studies.
• CRISPR works exceptionally well in yeast thanks to homologous recombination.
• Fluorescent dyes reveal mitochondria, vacuoles, nuclei, and damage.
• Lifespan assays (RLS & CLS) are core tools of aging research.
• Mitochondrial and vacuole stains uncover key aging phenotypes.
• Many advanced techniques require fluorescence microscopes or specialized equipment.
• Your home light microscope can still show budding, stress responses, and basic morphology.