Exam 3 Study Guide

Question 1

What are the major types of DNA mutations and how do they affect protein structure/function?

Answer 1

Point mutation: may cause missense, nonsense, or silent changes, altering protein shape/activity

Insertion/deletion (indel): may cause frameshift → truncated or dysfunctional protein

Large deletions/duplications: remove or add domains → loss/altered function

Inversions/translocations: disrupt gene integrity or regulatory context

Question 2

What are the components of the CRISPR–Cas9 system and what does each do?

Answer 2

Cas9: nuclease that cuts DNA

sgRNA: guides Cas9 to a complementary DNA target

PAM (NGG): sequence Cas9 must recognize to cut

Question 3

How do you design an sgRNA for a target sequence?

Answer 3

Choose a 20 nt sequence complementary to the target immediately upstream of a PAM (NGG), minimize off-target similarity, and ensure guide efficiency

Question 4

What is the evolutionary origin of CRISPR-Cas9 and how does it compare to restriction enzymes and RNAi?

Answer 4

CRISPR originated as a bacterial immune system using RNA-guided DNA cleavage

Restriction enzymes: recognize short DNA motifs; not programmable

RNAi: targets RNA for degradation; temporary, not genomic.
CRISPR is RNA-guided, programmable, and targets DNA

Question 5

What is the difference between in vitro and in vivo genome editing?

Answer 5

In vitro: in a test tube; shows Cas9 + sgRNA alone cut DNA

In vivo: in cells/organisms; depends on cellular repair pathways

Question 6

What part of editing is done by Cas9 and what is done by cellular repair pathways?

Answer 6

Cas9 supplies the double-stranded break
The cell supplies repair via NHEJ (indels) or HDR (precise edits)

Question 7

What are the DNA repair outcomes after Cas9 cuts?

Answer 7

NHEJ: fast, error-prone → indels → knockout

HDR: precise editing using donor template → knock-in

Question 8

What Cas9 variants allow labeling, upregulation, downregulation, or base editing?

Answer 8

dCas9: binds DNA without cutting → can regulate expression

dCas9 activation: attached to transcription activator protein

dCas9 inhibition: attached to gene repressor protein

Base editors (ABE, CBE): convert one nucleotide to another without DSB

Cas9-GFP: genomic imaging

Question 9

How can CRISPR create small indels, large deletions, or large insertions?

Answer 9

Small indels: single sgRNA + Cas9 → NHEJ

Large deletions: two guides flanking region → deletion of interval

Insertions: HDR template enables knock-in of genes/tags

Question 10

Give two examples of CRISPR-based therapies

Answer 10

Sickle-cell therapy: targeting BCL11A enhancer, with less BCL11A activity fetal hemoglobin increases, expression preventing sickling

Error of metabolism (baby KJ): use base editing to fix loss of fxn mutation in CPS-1 enzyme

Question 11

What obstacles prevent widespread CRISPR therapeutics?

Answer 11

Delivery problems, off-target effects, immune responses to Cas9, efficiency of HDR, ethical considerations

Question 12

Define proliferation, differentiation, potency, and stem cell

Answer 12

Proliferation: cell division

Differentiation: becoming specialized

Potency: range of possible fates (totipotent → multipotent)

Stem cell: self-renewing cell capable of differentiation

Question 13

How does differentiation occur during development?

Answer 13

Through changing transcription factor activity, chromatin states, and gene expression patterns that lock cells into specific fates

Question 14

Compare embryonic vs adult stem cells

Answer 14

Embryonic: pluripotent, broad potential

Adult: multipotent, tissue-specific functions

Question 15

What are iPS cells and how are they useful?

Answer 15

Reprogrammed adult cells made pluripotent using transcription factors
Used for disease modeling, personalized medicine, and potential therapies

Question 16

Why combine iPS therapy with CRISPR?

Answer 16

CRISPR can correct mutations, then iPS cells can be differentiated into healthy autologous tissues with no immune rejection

Question 17

What obstacles limit stem cell therapies?

Answer 17

Tumor formation risk, differentiation control, immune rejection, integration into tissues, ethical issues

Question 17

What are five mechanisms of eukaryotic gene regulation?

Answer 17

1. Epigenetic: DNA methylation, histone modification
2. Transcriptional: promoters, enhancers, TFs
3. RNA processing: splicing, 5’ cap, poly-A tail
4. Translation control: ribosome recruitment
5. Post-translational: phosphorylation, ubiquitination

Question 18

Define histone, enhancer, silencer, transcription factor, and splicing.

Answer 18

Histone: proteins packaging DNA

Enhancer: activates transcription via TF binding

Silencer: represses transcription

Transcription factor: protein that binds DNA to control transcription

Splicing: removing introns, combining exons

Question 19

How do transcription factors work in combination?

Answer 19

They bind regulatory DNA in distinct combinations that determine cell identity and gene expression patterns (combinatorial control)

Question 20

what are transgenes?

Answer 20

A gene from one organism that has been artificially introduced into the genome of another organism

Question 21

How do scientists use cloned regulatory elements to control transgenes?

Answer 21

By placing transgenes under specific promoters/enhancers, expression occurs only in certain tissues or developmental windows

Question 22

How are reporter genes used to study regulatory elements?

Answer 22

Regulatory sequences are fused to GFP/lacZ; if expressed, the reporter shows where and when the element functions

Question 23

What is RNAi and what stage of gene expression does it regulate?

Answer 23

Small RNAs (siRNA/miRNA) guide RISC to mRNA, causing degradation or translation inhibition—thus regulating post-transcriptional gene expression

Question 24

What are the uses and limitations of RNAi therapeutically?

Answer 24

Uses: silencing disease genes or viral RNA Limitations: off-target effects, delivery issues, temporary action

Question 25

What methods visualize or quantify RNA/protein?

Answer 25

FISH: spatial RNA RT-qPCR: RNA quantity RNA-seq: global transcriptome Western blot: protein quantity Immunofluorescence: protein location Use depends on whether you need location or amount

Question 26

Why do cells need to send and receive signals?

Answer 26

For coordination, homeostasis, responding to stimuli, controlling growth/metabolism, and neuronal communication

Question 27

Define ligand, agonist, antagonist, second messenger, synapse

Answer 27

Ligand: signaling molecule Agonist: activates receptor Antagonist: blocks receptor Second messenger: intracellular amplifier (ex. cAMP) Synapse: junction between signaling cells

Question 28

Compare ionotropic vs metabotropic neurotransmission

Answer 28

Ionotropic: ligand-gated ion channels, fast, direct ion flow Metabotropic: GPCRs, slower, uses second messengers

Question 29

How do signaling molecules work structurally and functionally?

Answer 29

Ligand binds receptor → conformational change → activates downstream proteins → triggers cellular response

Question 30

How do medicines activate or block signaling pathways?

Answer 30

They act as agonists (activate) or antagonists (block), altering receptor activation and downstream signaling

Question 31

Give examples of extracellular vs intracellular signals

Answer 31

Extracellular: hormones, neurotransmitters, growth factors Intracellular: Ca²⁺, cAMP, phosphorylation states

Question 32

How are extracellular signals converted to intracellular responses?

Answer 32

Receptor activation triggers ion flow (ionotropic) or G-protein cascades (GPCRs) → second messengers → cellular changes

Question 33

How can signaling change cellular behavior? Examples?

Answer 33

Changes in gene expression, metabolism, firing rate, contraction Examples: adrenaline increases heart rate; insulin changes glucose uptake; ACh slows heart

Question 34

What are consequences of dysregulated signaling?

Answer 34

Cancer (overactive RTKs), neurological disorders (imbalanced neurotransmission), metabolic disease (insulin resistance)