Exam 2 : Modules 5 - 9

Compare genome engineering and gene therapy.

• Genome engineering (gene editing):

  • Edits DNA by altering, removing, or adding specific nucleotides

  • Can occur at DNA, RNA, or epigenetic level

• Gene therapy:

  • Transfers whole genes using recombinant DNA

Key Difference:
👉 Genome engineering = precise edits to existing DNA
👉 Gene therapy = adds new genes

What are the steps of CRISPR-Cas9 genome editing?

1. Guide RNA (gRNA) is created to match a specific sequence of DNA

2. gRNA and Cas9 protein (restriction nuclease/enzyme) are added to the cell using vector

3. gRNA finds target sequence and Cas9 makes a double strand break (DSB)

4. Cell uses natural DNA repair mechanisms to fix the DSB

Why is DNA double-strand break repair critical?

• CRISPR works by creating a DSB in DNA

• The repair pathway determines the outcome

Two pathways:

• NHEJ:

  • Error-prone

  • Causes insertions/deletions (indels)

  • Often used for gene knockouts

• HDR (homologous recombination):

  • Uses template

  • Precise repair

👉 Control of repair = major challenge

How was CRISPR discovered and what’s its role?

• 1987: CRISPR sequences identified

• 2000–2005: Found in bacteria; spacers come from viruses

• 2006–2007: Identified as adaptive immune system

Function in bacteria:

1. Virus infects cell

2. Bacteria store viral DNA as spacers

3. On reinfection, CRISPR RNA guides enzymes to cut viral DNA

👉 Acts as acquired immunity in prokaryotes

What are the major technical challenges associated with CRISPR?

• Increasing specificity & efficiency

• Controlling DNA repair pathway

• Off-target effects

• Delivery issues

• HDR is inefficient in many cells

Problems with DSBs:

• Mixed/incorrect edits

• Translocations

• p53 activation → cancer risk

What ethical issues are associated with CRISPR?

• Autonomy: informed consent

• Religion: moral objections

• Risk vs. benefit: unintended consequences

• Fairness: access & inequality

• Regulation: who controls use

👉 Big concern: germline editing (heritable changes)

What are examples of genome editing applications?

• Muscle-bound dogs

• Bruise-resistant bananas

• Pig-to-human transplants

• Disease-resistant organisms

Adaptative Immunity

• Also called acquired immunity

• Uses specific antigens to target pathogens

• Activated after exposure to a pathogen

• Has immunological memory → stronger future responses

• Slower response than innate immunity

Base Editing

A newer genome editing approach uses the components from CRISPR systems together with other enzymes to directly install point mutations into cellular DNA or RNA without making double-stranded DNA breaks (DSBs).

Basic Science Research

• often called fundamental or bench research

• provides the foundation of knowledge for the applied science that follows

Cas9

• RNA-guided DNA-cutting enzyme (endonuclease)

• Part of CRISPR immune system

• Recognizes and cuts foreign DNA (e.g., viruses, plasmids)

Clinical Medicine

A field of medicine that deals primarily with the practice and study of medicine based on the direct examination of the patient.

CRISPR

• a family of DNA sequences in bacteria

• contains snippets of DNA from viruses that have attacked the bacterium

• snippets are used by the bacterium to detect and destroy DNA from further attacks by similar viruses

• sequences play a key role in a bacterial defense system

• sequences form the basis of a genome editing technology known as CRISPR/Cas9 that allows permanent modification of genes within organisms

Designer Babies

A human embryo that has been genetically modified, usually following guidelines set by the parent or scientist, to produce desirable traits.

DNA Repair

A collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome.

Gene Drives

• the phenomenon in which the inheritance of a particular gene or set of genes is favorably biased

• can arise through a variety of mechanisms

• results in its prevalence increasing in a population

Gene Editing

A type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of a living organism using engineered nucleases, or "molecular scissors."

Genome Engineering

A type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of a living organism using engineered nucleases, or "molecular scissors."

Homologous Recombination

• a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA

• most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks

NAS

National Academy of Sciences

Non-Homologous End Joining

• a pathway that repairs double-strand breaks in DNA

• referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template

• faster but mor error-prone than homologous recombination

Nucleases

An enzyme that cleaves the chains of nucleotides in nucleic acids into smaller units.

Prime Editing

• a ‘search-and-replace’ genome editing technology that directly writes the genetic information into a targeted DNA site

• uses modified Cas9 + reverse transcriptase + pegRNA

• writes new genetic information directly into DNA

Protospacer Adjacent Motif (PAM)

• a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system

• Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence

TALENs

Transcription Activator-Like Effector Nucleases are a class of engineered nuclease that can be used for gene editing.

Translational Research

Applies findings from basic science to enhance human health and well-being.

Zinc-Finger Nucleases (ZFNs)

A class of engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations.

Aneuploid

The presence of an abnormal number of chromosomes in a cell.

Autosomal Dominant

• One of several ways that a trait or disorder can be passed down (inherited) through families.

• In this type of disease, if you inherit the abnormal gene from only one parent, you can get the disease.

Autosomal Recessive

• One of several ways that a trait, disorder, or disease can be passed down through families.

• This type of disorder means two copies of an abnormal gene must be present in order for the disease or trait to develop.

Autosomes

Any chromosome that’s not a sex chromosome.

Crossing Over

The exchange of genetic material between homologous chromosomes that results in recombinant chromosomes during sexual reproduction.

Dominant Trait

A trait that will appear in the offspring if one of the parents contributes it.

Genotype

The genetic makeup of an organism or group of organisms with reference to a single trait, set of traits, or an entire complex of traits.

Incompletely Dominant

• A form of intermediate inheritance in which one allele for a specific trait is not completely expressed over its paired allele.

• This results in a third phenotype in which the expressed physical trait is a combination of the phenotypes of both alleles.

Microtubules

• Filamentous intracellular structures that are responsible for various kinds of movements in all eukaryotic cells.

• They’re involved in:

  • nucleic and cell division

  • organization of intracellular structure

  • intracellular transport

  • ciliary and flagellar motility

Non-Disjunction

The failure of one or more pairs of homologous chromosomes or sister chromatids to separate normally during nuclear division, usually resulting in an abnormal distribution of chromosomes in the daughter nuclei.

Pedigree

A diagram showing the lineage or genealogy of an individual and all the direct ancestors, usually to analyze or follow the inheritance of trait.

Phenotype

• An organism's expressed physical traits.

• It’s determined by an individual's genotype and expressed genes, random genetic variation, and environmental influences.

Recessive Trait

• An inherited trait that is outwardly obvious only when two copies of the gene for that trait are present.

• The condition is seen only in the absence of the dominant gene.

Sex Chromosome Disorders

A group of genetic conditions that are caused or affected by the loss, damage or addition of sex chromosomes (gonosomes).

Sex Chromosomes

• One pair of the total of 23 pairs of chromosomes.

• Individuals having two X chromosomes (XX) are female.

• Individuals having one X chromosome and one Y chromosome (XY) are male.

Sex Determination

A biological system that determines the development of sexual characteristics in an organism.

Spindle Apparatus

The cytoskeletal structure of eukaryotic cells that forms during cell division to separate sister chromatids between daughter cells.

Trisomy

A type of aneuploidy (an abnormal number of chromosomes), in which there are three instances of a particular chromosome, instead of the normal two.

X-Linked Dominant

A mode of genetic inheritance by which a dominant gene is carried on the X chromosome.

X-Linked Recessive

A mode of inheritance in which a mutation in a gene on the X chromosome causes the phenotype to be expressed in males (who are necessarily hemizygous for the gene mutation because they have one X and one Y chromosome) and in females who are homozygous for the gene mutation,

Describe how mutations lead to genetic diseases and disorders.

• Genetic disorders are diseases caused in whole or in part by changes in DNA sequence.

• Mutations can change protein function, localization, or amount, which leads to abnormal phenotypes (disease).

• Many Mendelian phenotypes result from altered protein function due to gene variants.

Explain Mendel’s Principle of Segregation.

• Each parent has two “factors” (alleles) for a trait.

• During gamete formation, one allele is passed to offspring with equal probability.

Describe the 3 characteristics of Mendelian Inheritance.

• Traits controlled by dominant and recessive alleles.

• Only two alleles are involved for a given gene.

• Phenotypic ratios can be predicted theoretically.

Describe Mendel’s crosses and the results of these crosses.

• Mendel used pure-breeding pea plants with traits in two forms.

  • This always produced offspring of the same phenotype when intercrossed.

• Parental cross → F1 generation showed only dominant phenotype (100%).

• Self-pollination of F1 → F2 showed 3:1 phenotype ratio (dominant : recessive).

Predict outcomes of a genetic cross using a Punnett square and probability.

To predict outcomes:

1. Identify gametes produced by each parent.

2. Draw a 2×2 grid.

3. Place the gametes from each parent on either side of the grid.

4. Determine the fertilization outcomes.

5. Determine the phenotype for each genotype.

Describe why a Punnett square cannot predict the probabilities of genotypes and phenotypes in a population.

• They predict expected theoretical ratios, not actual population frequencies.

• Real populations are influenced by environmental factors, chance, and complex inheritance patterns, so actual ratios may differ.

Describe how the heterozygote advantage allows for autosomal recessive diseases to be maintained in the population.

• Some recessive disease alleles remain common because heterozygotes gain survival benefits.

• Examples:

  • Cystic fibrosis carriers have increased resistance to dehydration from infections like cholera or typhoid.

  • Sickle-cell carriers have protection against malaria.

• This advantage increases allele frequency → disease persists in population.

What contributed to the success of Mendel’s crosses?

• Choosing characteristics that came in 2 forms

• Starting with pure-breeding strains

• Careful observation and meticulous documentation

What are the main patterns of inheritance?

• Autosomal dominant

• Autosomal recessive

• X-linked (sex-linked)

• Non-Mendelian (incomplete dominance, codominance, multiple alleles)

What is autosomal dominant inheritance?

• Only one copy of mutant allele needed

• Appears in every generation

• Affects males and females equally

• Example: Huntington’s disease

What is autosomal recessive inheritance?

• Requires two mutant alleles

• Can skip generations

• Parents often carriers

• Examples: Cystic fibrosis, Tay-Sachs, sickle cell anemia

What is X-linked recessive inheritance?

• Gene located on X chromosome

• Males more affected (only one X)

• Females usually carriers

• Fathers cannot pass to sons

• Examples:

  • Red-green color blindness

  • Hemophilia

  • Duchenne muscular dystrophy

How do you analyze a pedigree?

• Look for:

  • Pattern across generations

  • Whether trait skips generations

  • Male vs female frequency

• Use clues to determine inheritance type

How do you recognise autosomal dominant in a pedigree?

• Appears every generation

• Affected individuals have affected parent

• Equal in males and females

How do you recognise autosomal recessive in a pedigree?

• Can skip generations

• Unaffected parents can have affected child

• Equal in males and females

How do you recognise X-linked recessive in a pedigree?

• More males affected

• No father → son transmission

• Carrier mothers pass to sons

What are the basic pedigree symbols?

• Square = male

• Circle = female

• Filled = affected

• Half-filled = carrier

• Horizontal line = mating

• Vertical line = offspring

How do you determine genotype of individuals in a pedigree for autosomal dominant?

• Affected = AA or Aa

• Unaffected = aa

How do you determine genotype of individuals in a pedigree for autosomal recessive?

• Affected = aa

• Carrier = Aa

• Unaffected = AA

What determines biological sex in humans?

• XX = female

• XY = male

• Determined by genes on chromosomes

What is the SRY gene?

• Located on Y chromosome

• Triggers male development (testes formation)

What is the difference between sex and gender?

• Sex = biological (chromosomes, anatomy)

• Gender = social/cultural identity

What is androgen insensitivity syndrome (AIS)?

• XY individual

• Produces testosterone but cells cannot respond

• Develops female phenotype

What’s the key idea about sex determination systems?

• Not all organisms use XY

• Other systems:

  • XX/XO

  • ZZ/ZW

  • Haplodiploid (bees)

What does “lack of dominance” mean?

• Neither allele fully dominates the other

• Leads to non-Mendelian patterns

What is incomplete dominance?

• Heterozygote shows intermediate phenotype

• Example:

  • Red × White → Pink flowers

  • Black × White chickens → Blue

What is the typical ratio in incomplete dominance?

• Genotype: 1:2:1

• Phenotype: 1:2:1 (NOT 3:1)

What is codominance?

• Both alleles fully expressed

• No blending

• Example:

  • Black + white chicken → speckled feathers

What are multiple alleles?

• More than 2 alleles exist in population

• Individual still inherits only 2

• Example: ABO blood types (A, B, O)

Compare incomplete dominance vs. codominance vs. multiple alleles.

• Incomplete dominance → blended phenotype

• Codominance → both traits visible

• Multiple alleles → more than 2 allele options exist

How do Punnett squares work for incomplete dominance?

• Show genotype ratios (1:2:1)

• Phenotypes follow same ratio

• Example: CRCR × CWCW → all CRCW (pink)

How do Punnett squares work for codominance?

• Heterozygote shows both traits

• Ratios still 1:2:1

How do Punnett squares work for multiple alleles?

• Use more allele options

• Follow dominance relationships (e.g., A and B codominant, O recessive)

What is nondisjunction?

• Failure of chromosomes to separate properly during meiosis

• Leads to abnormal number of chromosomes (aneuploidy)

What gametes are produced from nondisjunction?

• Gametes may have:

  • n + 1 (extra chromosome)

  • n – 1 (missing chromosome)

• Can occur in meiosis I or II

What is aneuploidy?

• Abnormal number of chromosomes

• Example: 45 or 47 instead of 46

What is trisomy?

• Extra chromosome (3 copies)

• Example: Down syndrome (Trisomy 21)

What is monosomy?

• Missing one chromosome from a pair

• Only viable example in humans: X chromosome

What are structural chromosomal abnormalities?

• Deletion (missing segment)

• Duplication (extra segment)

• Translocation (segment moved)

• Inversion (segment flipped)

How do you predict outcomes of a genetic cross?

• Use Punnett squares

• Identify parental genotypes

• Determine possible gametes

• Combine to find offspring ratios

How do you calculate probability of a genetic outcome?

• Use Punnett square ratios

• Multiply probabilities for independent events

• Example:

  • 1/2 × 1/2 = 1/4

How do nondisjunction events affect genetic crosses?

• Introduce abnormal chromosome numbers

• Leads to conditions like trisomy or monosomy

What skills are needed for genetic case studies?

• Identify inheritance pattern

• Use pedigrees

• Calculate probabilities

• Recognize chromosomal abnormalities

What is a Mendelian trait/disease?

• Controlled by one gene

• Large effect size

• Clear inheritance pattern

• Example: APOB mutation causing high cholesterol

What is a polygenic trait/disease?

• Controlled by many genes

• Each gene has small effect

• Influenced by environment

• Most traits/diseases are complex

What is a quantitative trait?

• Continuous variation

• Shows bell-shaped distribution

• Example: cholesterol levels

What is a discrete trait?

• Distinct categories (yes/no)

• Example: disease presence (hypercholesterolemia)

What are examples of polygenic traits/disease?

• Asthma

• Diabetes

• Heart disease

• Obesity

• Autism

• Hypertension

What is a key feature of polygenic traits?

• Many genetic variants contribute

• Effects add together

• No single gene determines outcome

What is a polygenic risk score?

• Combines effects of many genetic variants

• Estimates individual risk for a trait/disease

• Based on cumulative genetic contribution (effect sizes)

What is heritability?

• Measure of how much variation in a trait is due to genetics

• Range: 0 → 1

  • 0 = environment only

  • 1 = genetics only

What is a complex trait/disease?

• Influenced by:

  • Multiple genes

  • Environment

  • Lifestyle

• Also called multifactorial

How do lifestyle and environment influence traits?

• Can shift phenotype distribution

• Interact with genetic variants

• Examples:

  • Diet → cholesterol levels

  • Exercise → heart disease risk

  • Environment → disease expression