Exam 3 - Foundations in Genetics (4.16.26)

What is the biological function of CRISPR in bacteria?

Adaptive immune system that protects against phage infection by cutting foreign DNA

What does the CRISPR locus contain?

Repeated sequences + spacer DNA from past phage infections (a “genetic memory”)

What protein is essential for CRISPR DNA cutting?

Cas9

What RNA molecules guide Cas9?

crRNA + tracrRNA (can be fused into gRNA)

What is a gRNA (guide RNA)?

A synthetic RNA combining crRNA + tracrRNA that directs Cas9 to a target DNA sequence

What must be present near the target DNA for Cas9 to cut?

PAM site (e.g., NGG)

Where does Cas9 cut relative to PAM?

~3 base pairs upstream of the PAM site

What type of DNA break does CRISPR create?

Double-strand break (DSB)

What are the two main DNA repair pathways after CRISPR cutting?

1. NHEJ (Non-homologous end joining)

2. HDR (Homology-directed repair)

What is non-homologous end joining (NHEJ)?

a primary, often error-prone pathway for repairing DNA double-strand breaks (DSBs) by directly ligating broken ends without a homologous template

What does NHEJ do?

Error-prone repair → insertions/deletions (knockouts)

What is homology directed repair (HDR)?

Precise mechanism used by cells to repair DNA double-strand breaks (DSBs) by utilizing a homologous template,

What does homology directed repair (HDR) do?

Uses a donor template for precise edits

What can CRISPR be used for?

1. Gene knockouts

2. Point mutations

3. insertions (transgenes)

4. Gene corrections

What are the 3 key components for CRISPR editing?

1. Cas9 protein

2. gRNA

3. (optional) Repair template for HDR

What is CRISPRa?

Activates gene expression using dead Cas9 + activators

What is CRISPRi?

Represses gene expression (interference)

What are CRISPR screens?

high-throughput, genome-wide studies are used to identify genes responsible for specific phenotypes, such as drug resistance or disease progression, by using CRISPR-Cas9

How can CRISPR components be delivered into cells?

1. Plasmids

2. RNPs (protein + RNA)

What are the core components of a virus?

1. Genome (DNA / RNA)

2. Capsid

3. Surface Proteins (for entry)

4. (sometimes) Envelope

What is a capsid?

the rigid, protective protein shell of a virus particle that surrounds and encloses its nucleic acid genome (DNA or RNA)

What is the difference between enveloped and non-enveloped viruses?

• Enveloped: lipid membrane (e.g, HIV)

• Non-enveloped: no membrane (e.g, adenovirus)

What types of genomes can viruses have?

• DNA or RNA

• Single-stranded or double-stranded

Why are viral genomes small?

They rely on host machinery and are highly specialized.

• small genome allows for faster replication

Steps of viral replication cycle?

1. Viral entry

2. Replication

3. Assembly into Viral Particles

4. Egress/Exit

5. New Infection Cycle

What is special about retroviruses?

They integrate DNA into the host genome

• use the enzyme reverse transcriptase to convert their RNA genome into DNA within a host cell

What is a risk of viral integration?

Insertion into oncogenes/tumor suppressors → cancer

What is gene therapy?

Treating disease by modifying a patient’s DNA

• introduction of WT copies of a gene

What diseases are easiest to treat with gene therapy?

Autosomal recessive (loss-of-function mutations)

Why are viruses used in gene therapy?

They naturally deliver DNA into cells

Common viral vectors?

• Adenovirus

• Retrovirus / Lentivirus

• AAV (adeno-associated virus)

Which vector is best for in-vivo?

Adeno-associated virus (AAV)

Which vector is best for ex-vivo?

Retro/Lenti-virus

Which viral vector integrates into genome?

Retrovirus/lentivirus

Which vector carries the largest DNA?

Adenovirus (~35kb)

What features are important for all gene delivery vectors?

• avoid disruption of other genes

• expression of delivered gene

• size of DNA/gene

• cost

What is AAV?

A small, safe viral vector commonly used in gene therapy

• no disease, but can still infect humans.

• ssDNA

What limits AAV?

Small cargo size (~4.7 kb)

Difference between in vivo and ex vivo gene therapy?

• In vivo: deliver directly into patient

• Ex vivo: modify cells outside body, then reinsert

How does CRISPR achieve specificity?

gRNA base pairing + PAM requirement

What determines where CRISPR cuts?

gRNA sequence

What determines edit outcome?

DNA repair pathway (NHEJ v. HDR)

Biggest risk of viral gene therapy?

Random integration → cancer

What is a phage?

a virus that specifically infects, replicates within, and destroys bacteria

What is a retro/lentivirus

Multiple diseases (HIV/AIDS)

• ssRNA

What is a adenovirus?

a respiratory illnesses (colds)

• dsDNA

What is an inverted terminal repeats?

non-coding sequence. determines what gets packed into virus

At what levels can gene regulation occur?

Replication, transcription, RNA processing, translation, and posttranslational modification.

How do post-translational modifications affect stability/function of proteins?

They are chemical changes made to a protein after it’s translated, and they can drastically change both how long the protein lasts (stability) and what it does (function).

• phosphorylation, cleavage, methylation, acetylation, ubiqutiation

What is the main reason cell-specific protein expression happens?

Strict regulation of transcription at different locuses.

• alcohol dehydrogenase gene turned on in liver cell; not neuron cells

• neurotransmitter gene turned on in neuron cell’s locus; not liver cells

What is a eukaryotic transcription unit?

segment of DNA—typically a single gene—transcribed into a single pre-mRNA or RNA molecule

• A region containing a promoter, RNA-coding region, and terminator.

What is the promoter?

A DNA region where RNA polymerase and transcription machinery assemble to begin transcription.

• have many different transcription factor binding sites

What is the TATA box?

A common core promoter sequence involved in transcription initiation.

• on the coding sequence

What are transcription factors?

DNA-binding proteins that regulate transcription.

• are all DNA binding proteins at DNA binding domain

What is the core promoter?

The minimal DNA region required to start transcription.

What is the regulatory promoter?

DNA regions upstream of the core promoter that control how much transcription occurs.

• bound by specialized TFs

What does it mean that transcription factor (TF) binding sites are “mixed and matched” in promoters?

Promoters contain different combinations of TF binding sites, and each unique combination of transcription factors determines the level and specificity of gene expression (combinatorial control).

What does it mean that a single transcription factor can bind many different promoters?

One transcription factor can bind to multiple promoters across the genome, allowing it to regulate the expression of many different genes.

What do activators do?

They increase transcription by helping transcription machinery assemble or function.

• Turn on or up transcription by binding to DNA (helps recruit TFs and RNA pol)

What do repressors do?

They decrease or block transcription.

• inhibit transcription by blocking proper assembly of RNA pol. on the core promoter

What are enhancers?

Regulatory DNA sequences that increase transcription, often from far away and at distant locations in genome

Why can enhancers work even when far from a gene?

Because DNA loops in 3D space, bringing enhancers close to promoters.

What are mediators in gene regulation?

large protein complexes that act as a bridge between transcription factors and RNA polymerase, helping transmit regulatory signals to initiate or regulate transcription.

• works directly with enhancers to regulate gene transcription, serving as a critical bridge

• facilitates DNA looping

What does it mean that enhancers can be tissue-specific?

They can drive gene expression in only certain cell types or tissues.

How important is the proximity of DNA?

Closeness is key, however DNA is not linear; therefore factors sequences can be far apart on a sequence but can be close spacially in 3D space, so it can still interact

What is the Tbx4 gene?

A transcription factor gene involved in hindlimb and lung development in mice.

• bound by TFs thar are made in diff. parts of the body and at diff. times.

What role does Tbx4 play in development?

It regulates genes required for proper formation of hind legs and lungs.

Where is Tbx4 expressed?

In tissues that will become the hindlimbs and lungs.

What is a reporter gene?

A gene used to visualize or measure gene expression, often producing an easily detectable signal (like fluorescence).

Why are reporter genes important in studying Tbx4?

They allow researchers to see where and when Tbx4 regulatory elements are active.

How is transcription regulation a combinatorial process?

May have several binding sites for activators and repressors; therefore gene can only be expressed if the right combination.

• both activators = strong txn.

• only one activator = less txn.

• both activators + a repressor = no txn.

What is combinatorial regulation?

Gene expression depends on the combination of activators and repressors bound to DNA.

What are GAL genes in yeast used for?

They are involved in galactose utilization.

• used as an example of gene regulation in yeast

How do glucose and galactose affect GAL gene expression?

Glucose represses GAL genes, while galactose activates them.

What does GAL1 encode?

Galactokinase, the first enzyme in galactose utilization.

How is viewing + / - presence of galactose with a WT and mutant of GAL1 useful?

You can use it as a readout for GAL gene activation (GAL1 = reporter gene)

• Gal1+ (WT) = growth only in +galactose

• Gal1- (Mutant) = no growth in -Gal and +Galactose

How is GAl1 a reporter gene / readout?

Genetic screens can be used to see what couldn’t grow on galactose (presence = [+] Galactose, absence [-] galactose)

What is epistasis analysis used for in regulatory pathways?

To determine the order and function of genes in a pathway.

What does constitutive mean?

the study of genes that are constantly transcribed and expressed ("always on") in a cell, regardless of environmental conditions

How to confirm a correct epistatic pathway?

Run a double mutant; and the phenotype will tell you which is the epistatic gene

What is an epistatic gene?

gene that masks or overrides the effect of another gene in a pathway. In epistasis analysis, the gene whose phenotype is seen is typically downstream, meaning it acts later in the pathway and determines the final outcome.

What does uninducible mean in gene regulation?

a gene cannot be turned on (activated) even when the proper signal or inducer is present.

What is the GAL4/UAS system?

A gene expression system where GAL4 binds UAS sequences to activate a target gene.

• controls gene expression in other organisms; (e.g, uses a yeast transcription factor to control gene expression in specific tissues of another organism, like flies)

Why is the GAL4/UAS system useful experimentally?

It allows scientists to control gene expression in specific tissues or cell types.

What is the Upstream Activating Sequence (UAS)?

cis-acting regulatory DNA segment, primarily in yeast, that increases the expression of adjacent genes by binding specific transcriptional activators

What is a GAL4 driver line?

a transgenic organism (usually a fruit fly, Drosophila) that expresses a specific transcriptional activator—most commonly GAL4—in particular tissues or cells.

• tissue cell-type specific expression

What happens during RNA splicing?

Introns are removed and exons are joined to form mature mRNA.

What is alternative splicing?

Different combinations of exons are joined to create different mRNAs from one gene.

Why is alternative splicing important?

It increases protein diversity without increasing gene number.

• from a single-coding sequence

• exons 1,2,3,4,5 → [1] 1,3,5 | [2] 2,3,4 | [3] = 1,2,5

How can one gene produce many different proteins across different cells?

Through alternative splicing.

What is the alt. splicing example used?

Rat alpha-tropomyosin

What is Rat alpha-tropomyosin

a protein encoded by the Tpm1 gene that regulates actin-myosin interaction in striated and smooth muscles

• Through alternative splicing, this single gene produces multiple distinct mRNA isoforms, generating at least six different protein isoforms for striated, smooth, and nonmuscle cells (brain, fibroblasts) by mixing and matching exons

What are mRNA isoforms?

distinct mature mRNA molecules generated from a single gene locus through alternative splicing, different transcription start sites (TSS), etc.

What is spinal muscular atrophy (SMA)?

An autosomal recessive neuromuscular disease that weakens muscles, giving difficulty breathing, swallowing, etc.

• 1 in 15,000 births

• primarily caused by the loss of function of SMN1

What genes are important in SMA?

SMN1 and SMN2.

How do SMN1 and SMN2 differ functionally?

SMN1 mostly produces functional protein, while SMN2 often undergoes splicing that makes mostly nonfunctional protein but still some functional

• SMN2 viewed as the backup

Why is SMN2 called a “backup” gene in SMA?

because it can still produce some functional SMN protein (10% functional)

• has the exon 7 spliced out 90% of the time (which is key in producing func. protein)

• SMN1 has exon 7