• Gene editing allows highly specific changes to the genome.
• A major advance came from the CRISPR-Cas system, especially CRISPR-Cas9.
• In bacteria, CRISPR-Cas acts as a form of acquired immunity against:
  • phages
  • plasmids
• Bacteria capture pieces of invading DNA and incorporate them into their own genome as CRISPR sequences.
• These CRISPR sequences are then:
  • transcribed into RNA
  • processed into guide-like RNA
  • used to direct Cas9 nuclease to matching foreign DNA
• Cas9 then cuts the matching DNA → destroys the invading phage DNA.
• Gene editing uses the same principle with an artificial guide RNA (gRNA).
• The gRNA is designed to be complementary to a chosen DNA sequence.
• It binds Cas9 and guides it to the target DNA.
• At the target:
  gRNA + Cas9 → precise recognition → double-stranded DNA break (Fig. 4.4).
• The broken DNA can then be repaired in two important ways:
  • Nonhomologous end joining (NHEJ):
    • repairs the break directly
    • may introduce somewhat random disruptive mutations
    • therefore can disrupt the targeted gene
  • Homologous recombination:
    • allows precise introduction of a desired new DNA sequence
• Both the gRNA and Cas9 can be delivered into cells using a single plasmid.
• CRISPR-Cas9 is especially useful because of its:
  • high specificity
  • flexibility
  • relative ease of use
• Important applications include:
  • introducing specific mutations into cells to model cancer and other diseases
  • rapidly producing transgenic animals
  • potentially correcting mutations responsible for heritable diseases
• The same technology could also be used to alter less desirable traits, which has created major ethical debate about its use.

KEY CONCEPT

• CRISPR = targeting system
• gRNA = finds the matching DNA
• Cas9 = cuts the DNA
• After cutting:
  • NHEJ → disruptive mutation
  • Homologous recombination → precise DNA correction/insertion

CONCEPTUAL EXAMPLES

• Disease-causing DNA sequence identified → matching gRNA designed → Cas9 cuts it → targeted gene editing.
• Cas9 cut repaired by NHEJ → gene becomes disrupted → gene knockout-like effect.
• Cas9 cut repaired using a desired DNA template → specific sequence inserted or corrected → precise gene correction.

FIG. 4.4 — CRISPR/Cas9 Gene Editing

🧠 Simplest idea

gRNA finds the exact DNA target → Cas9 cuts both DNA strands → cell repairs the break → repair can create either a random mutation or a precise mutation.

Whole figure in one line

gRNA + Cas9 → target DNA recognition → double-stranded DNA break → NHEJ = random indel OR HDR + donor DNA = specific edit

1️⃣ TOP — gRNA guides Cas9

🟣 Purple strand = gRNA (guide RNA)
🟢 Large green structure = Cas9 protein
🔵 Double helix = normal double-stranded DNA

The gRNA contains a sequence homologous/complementary to the target DNA.

Arrow concept:

gRNA sequence matches target DNA
→ guides Cas9 to the correct genomic location.

🧠 Think:

gRNA = GPS
Cas9 = molecular scissors

2️⃣ Target genomic sequence

🔴 Red DNA region = the specific DNA sequence being targeted.

The complementary part of gRNA pairs with this target.

So:

Correct match → Cas9 positioned at the target

3️⃣ Cleavage

Black arrows labeled cleavage show Cas9 cutting:

• one DNA strand
• the opposite DNA strand

⬇️

Double-stranded DNA break

Meaning: both strands of DNA are cut.

After the break, TWO repair pathways are possible

4️⃣ LEFT — NHEJ

NHEJ = Non-Homologous End Joining

The broken DNA ends are simply joined back together.

But this repair is error-prone.

⬇️ Black arrow

Small DNA bases may be:

• inserted
• deleted

= Insertion / deletion (indel)

🟢/teal changed segment = random altered DNA.

Result:

DNA with random mutation

🧠 Easy idea:

NHEJ = quick repair, but messy

Often useful for knocking out/disrupting a gene.

5️⃣ RIGHT — HDR

HDR = Homology-Directed Repair

Here the cell is given:

🟡 Donor DNA

This donor DNA contains the desired new sequence.

The cell uses it as a template.

⬇️

Broken DNA + donor DNA template
→ accurate repair
→ desired sequence copied into chromosome.

Result:

DNA with specific mutation

🧠 Easy idea:

HDR = repair using a blueprint

🎨 COLOR / STRUCTURE GUIDE

• 🟢 Large green shape = Cas9 protein
• 🟣 Purple loop/strand = gRNA
• 🔵 DNA = surrounding genomic DNA
• 🔴 DNA segment = targeted/cut region
• 🟡 DNA segment = donor DNA carrying desired change
• 🟢/teal small altered region after NHEJ = random insertion/deletion
• ⚫ Black arrows = repair pathway direction
• 🟨 Yellow arrows = Cas9/gRNA action and targeting

⭐ MAIN DIFFERENCE

NHEJ

No donor template
→ inaccurate repair
→ insertion/deletion
→ random mutation

HDR

Donor DNA provided
→ template-guided repair
→ specific mutation

🧠 Fastest exam recall

gRNA finds → Cas9 cuts → cell repairs.

NHEJ = random indels
HDR = precise edit using donor DNA

⭐ One-line memory

CRISPR = GPS (gRNA) + scissors (Cas9) + repair (NHEJ or HDR).

GENETIC DISEASES

• Three commonly used terms should be distinguished:
  • Hereditary
  • Familial
  • Congenital
• Hereditary disorders:
  • are transmitted through the parents’ gametes
  • therefore, they are familial
• Congenital simply means present at birth.
• A congenital disease does not have to be genetic.
  • Example: congenital syphilis.
• Likewise, a genetic disease does not have to be congenital.
  • Example: Huntington disease is genetic, but symptoms usually begin in the third or fourth decade of life.

KEY CONCEPT

• Hereditary → transmitted through parental gametes → familial.
• Congenital → present at birth, but may or may not be genetic.
• Genetic disease → may appear at birth or much later in life.

CONCEPTUAL EXAMPLES

• Congenital syphilis → present at birth but not genetic.
• Huntington disease → genetic but usually not apparent at birth.

NATURE OF GENETIC ABNORMALITIES CONTRIBUTING TO HUMAN DISEASE

• Genetic abnormalities can alter the structure or function of proteins → disturb cellular homeostasis → cause disease.

Mutations in Protein-Coding Genes

• Mutation = permanent change in DNA.
• Germ-cell mutations → can be transmitted to offspring → may cause inherited disease.
• Somatic-cell mutations → are not inherited, but are important in cancers and some congenital disorders.
• Important mutation types include:
  • Point mutation = substitution of one nucleotide base by another.
    • It may replace one amino acid with another → missense mutation.
    • Example: mutation in the β-globin gene → hemoglobin S instead of hemoglobin A → sickle cell disease.
    • If the mutation creates a premature stop codon → shortened protein or failure of protein production → nonsense mutation.
  • Frameshift mutation:
    • insertion or deletion of 1 or 2 base pairs
    • shifts the DNA reading frame → abnormal downstream protein sequence.
  • Trinucleotide repeat mutation:
    • abnormal expansion of a 3-nucleotide sequence.
    • Example: fragile X syndrome → expansion of CGG repeats in the FMR1 gene.
    • Normal individuals average about 29 repeats.
    • Affected individuals may have about 200–4000 repeats.
    • Expansion can suppress normal FMR1 expression → intellectual disability.
    • These mutations are dynamic → repeat number may increase during gametogenesis.

Alterations in Protein-Coding Genes Other Than Mutations

• Protein-coding genes can also be altered structurally without simple sequence mutations.
• Important changes include:
  • amplifications
  • deletions
  • translocations
• These changes can produce abnormal gain or loss of protein function.
• They may occur:
  • in the germline
  • or be acquired in somatic cells.
• Some inherited abnormalities involve a whole chromosomal region rather than one gene.
  • Example: 22q microdeletion syndrome.
• Copy number variations have been linked with increased risk of disorders such as autism.
• Cancers commonly acquire:
  • amplifications
  • deletions
  • translocations
• Classic example:
  t(9;22) BCR-ABL translocation → Philadelphia chromosome → chronic myeloid leukemia.
• Genetic disorders can be divided into four major categories:
  • Mendelian disorders
    • mutation in a single gene
    • usually high penetrance
    • inherited and familial
    • include enzyme defects and other inborn errors of metabolism
  • Complex or multifactorial disorders
    • caused by multiple genes + environmental influences
    • examples: hypertension, diabetes, allergic diseases, autoimmune diseases
  • Chromosomal disorders
    • caused by abnormal chromosome number or structure
    • example: Down syndrome
  • Single-gene disorders with nonclassic inheritance
    • include:
      • triplet-repeat disorders
      • mitochondrial DNA mutations
      • disorders influenced by genomic imprinting

MENDELIAN DISORDERS: DISEASES CAUSED BY MUTATIONS IN SINGLE GENES

• Single-gene mutations usually follow classic Mendelian inheritance patterns (Tables 4.1 and 4.2).
• Main inheritance patterns:
  • autosomal dominant
  • autosomal recessive
  • X-linked
• Although individual Mendelian disorders are rare, together they cause an important disease burden.
• A single mutation can produce many different effects → pleiotropy.
  • Example: Marfan syndrome
  • Mutation in fibrillin → abnormalities in:
    • skeleton
    • eyes
    • cardiovascular system
• Different mutations in different genes can produce the same phenotype → genetic heterogeneity.
  • Example: different mutations can cause retinitis pigmentosa.
• Genetic heterogeneity is important for:
  • genetic counseling
  • molecular diagnosis
• The severity of a single-gene disorder may also be changed by other genes called modifier genes.
• Prenatal genetic screening in high-risk populations has reduced the incidence of some inherited diseases, such as Tay-Sachs disease (Table 4.1).

KEY CONCEPT

• Mutation = permanent DNA change.
• Germline mutation → inherited; somatic mutation → not inherited.
• Missense = wrong amino acid.
• Nonsense = premature stop.
• Frameshift = reading frame shifted.
• Trinucleotide repeat = repeated 3-base sequence expands.
• Major genetic disease groups = Mendelian + multifactorial + chromosomal + nonclassic single-gene disorders.
• Pleiotropy = one gene → many effects.
• Genetic heterogeneity = different genes → similar disease.
• Modifier genes = alter severity of disease.

CONCEPTUAL EXAMPLES

• β-globin point mutation → HbS → sickle cell disease.
• FMR1 CGG repeat expansion → gene silencing → fragile X syndrome.
• Fibrillin mutation → eye + skeletal + cardiovascular abnormalities → pleiotropy.
• Different gene mutations → same retinal disease → genetic heterogeneity.
• BCR-ABL translocation → Philadelphia chromosome → chronic myeloid leukemia.

NATURE OF GENETIC ABNORMALITIES CONTRIBUTING TO HUMAN DISEASE

• Genetic abnormalities can alter the structure or function of proteins → disturb cellular homeostasis → cause disease.

Mutations in Protein-Coding Genes

• Mutation = permanent change in DNA.
• Germ-cell mutations → can be transmitted to offspring → may cause inherited disease.
• Somatic-cell mutations → are not inherited, but are important in cancers and some congenital disorders.
• Important mutation types include:
  • Point mutation = substitution of one nucleotide base by another.
    • It may replace one amino acid with another → missense mutation.
    • Example: mutation in the β-globin gene → hemoglobin S instead of hemoglobin A → sickle cell disease.
    • If the mutation creates a premature stop codon → shortened protein or failure of protein production → nonsense mutation.
  • Frameshift mutation:
    • insertion or deletion of 1 or 2 base pairs
    • shifts the DNA reading frame → abnormal downstream protein sequence.
  • Trinucleotide repeat mutation:
    • abnormal expansion of a 3-nucleotide sequence.
    • Example: fragile X syndrome → expansion of CGG repeats in the FMR1 gene.
    • Normal individuals average about 29 repeats.
    • Affected individuals may have about 200–4000 repeats.
    • Expansion can suppress normal FMR1 expression → intellectual disability.
    • These mutations are dynamic → repeat number may increase during gametogenesis.

Alterations in Protein-Coding Genes Other Than Mutations

• Protein-coding genes can also be altered structurally without simple sequence mutations.
• Important changes include:
  • amplifications
  • deletions
  • translocations
• These changes can produce abnormal gain or loss of protein function.
• They may occur:
  • in the germline
  • or be acquired in somatic cells.
• Some inherited abnormalities involve a whole chromosomal region rather than one gene.
  • Example: 22q microdeletion syndrome.
• Copy number variations have been linked with increased risk of disorders such as autism.
• Cancers commonly acquire:
  • amplifications
  • deletions
  • translocations
• Classic example:
  t(9;22) BCR-ABL translocation → Philadelphia chromosome → chronic myeloid leukemia.
• Genetic disorders can be divided into four major categories:
  • Mendelian disorders
    • mutation in a single gene
    • usually high penetrance
    • inherited and familial
    • include enzyme defects and other inborn errors of metabolism
  • Complex or multifactorial disorders
    • caused by multiple genes + environmental influences
    • examples: hypertension, diabetes, allergic diseases, autoimmune diseases
  • Chromosomal disorders
    • caused by abnormal chromosome number or structure
    • example: Down syndrome
  • Single-gene disorders with nonclassic inheritance
    • include:
      • triplet-repeat disorders
      • mitochondrial DNA mutations
      • disorders influenced by genomic imprinting

MENDELIAN DISORDERS: DISEASES CAUSED BY MUTATIONS IN SINGLE GENES

• Single-gene mutations usually follow classic Mendelian inheritance patterns (Tables 4.1 and 4.2).
• Main inheritance patterns:
  • autosomal dominant
  • autosomal recessive
  • X-linked
• Although individual Mendelian disorders are rare, together they cause an important disease burden.
• A single mutation can produce many different effects → pleiotropy.
  • Example: Marfan syndrome
  • Mutation in fibrillin → abnormalities in:
    • skeleton
    • eyes
    • cardiovascular system
• Different mutations in different genes can produce the same phenotype → genetic heterogeneity.
  • Example: different mutations can cause retinitis pigmentosa.
• Genetic heterogeneity is important for:
  • genetic counseling
  • molecular diagnosis
• The severity of a single-gene disorder may also be changed by other genes called modifier genes.
• Prenatal genetic screening in high-risk populations has reduced the incidence of some inherited diseases, such as Tay-Sachs disease (Table 4.1).

KEY CONCEPT

• Mutation = permanent DNA change.
• Germline mutation → inherited; somatic mutation → not inherited.
• Missense = wrong amino acid.
• Nonsense = premature stop.
• Frameshift = reading frame shifted.
• Trinucleotide repeat = repeated 3-base sequence expands.
• Major genetic disease groups = Mendelian + multifactorial + chromosomal + nonclassic single-gene disorders.
• Pleiotropy = one gene → many effects.
• Genetic heterogeneity = different genes → similar disease.
• Modifier genes = alter severity of disease.

CONCEPTUAL EXAMPLES

• β-globin point mutation → HbS → sickle cell disease.
• FMR1 CGG repeat expansion → gene silencing → fragile X syndrome.
• Fibrillin mutation → eye + skeletal + cardiovascular abnormalities → pleiotropy.
• Different gene mutations → same retinal disease → genetic heterogeneity.
• BCR-ABL translocation → Philadelphia chromosome → chronic myeloid leukemia.

Transmission Patterns of Single-Gene Disorders

Disorders of Autosomal Dominant Inheritance

• Autosomal dominant disorders appear in the heterozygous state → only one mutant allele is enough to cause disease.
• Usually, at least one parent is affected.
• Males and females are affected equally, and both can transmit the disorder.
• If an affected person has children with an unaffected person:
  • each child has a 50% chance of inheriting the disease.
• Some affected patients have unaffected parents because the disorder results from a new mutation in the egg or sperm.
  • Their siblings are usually unaffected.
• Clinical expression can vary because of:
  • Reduced penetrance
    • person inherits the mutant gene but shows no clinical features.
  • Variable expressivity
    • everyone with the mutant gene shows the trait, but severity differs.
    • Example: neurofibromatosis type 1 may range from skin spots to multiple tumors and skeletal deformities.
• Some autosomal dominant diseases have delayed onset.
  • Examples:
    • Huntington disease
    • inherited mutations that increase risk of adult cancers
• In many autosomal dominant disorders, about 50% reduction of normal gene product is enough to cause disease.
• Because half the normal amount of many enzymes is usually sufficient, autosomal dominant disorders often involve proteins other than enzymes, especially:
  • Regulatory proteins
    • receptors or transport proteins
    • Example: LDL receptor mutation → familial hypercholesterolemia
  • Structural proteins
    • Example: collagen
    • Example: spectrin → hereditary spherocytosis
• Sometimes the mutant protein interferes with the normal protein → dominant-negative effect.
• Example:
  • Collagen normally consists of three chains forming a trimer.
  • A mutant collagen chain interferes with assembly of normal chains → marked reduction in functional collagen.
  • This occurs in some forms of osteogenesis imperfecta.

Disorders of Autosomal Recessive Inheritance

• Autosomal recessive disorders appear in the homozygous state → both alleles must be mutated.
• Typical pattern:
  • parents are usually unaffected carriers
  • several siblings may be affected
  • each child has a 25% chance of being affected
• If the mutant gene is rare, affected patients are more likely to have consanguineous parents.
• Autosomal recessive disorders form the largest group of Mendelian disorders.
• Compared with autosomal dominant disorders:
  • clinical expression is usually more uniform
  • complete penetrance is common
  • onset is often early in life
• New recessive mutations may occur, but they are often not noticed immediately because:
  • a heterozygous carrier is usually asymptomatic
  • the mutation may pass through several generations before two carriers have an affected child
• In many autosomal recessive disorders, the affected gene encodes an enzyme.
• Heterozygous carriers produce about:
  • 50% normal enzyme
  • 50% abnormal enzyme
• Usually, 50% normal enzyme activity is enough for normal cell function.
• Therefore, carriers usually remain clinically normal.

KEY CONCEPT

• Autosomal dominant = one mutant allele is enough → 50% risk to each child.
• Autosomal recessive = two mutant alleles required → 25% risk when both parents are carriers.
• Reduced penetrance = gene present, disease not expressed.
• Variable expressivity = same gene, different severity.
• Dominant-negative mutation = mutant protein interferes with normal protein.
• Autosomal recessive disorders commonly involve enzymes.

CONCEPTUAL EXAMPLES

• Affected parent + unaffected parent → each child has 1 in 2 chance → autosomal dominant.
• Two carrier parents → each child has 1 in 4 chance of disease → autosomal recessive.
• NF1 mutation → mild skin spots in one patient but tumors and skeletal changes in another → variable expressivity.
• Mutant collagen chain damages the function of normal collagen chains → dominant-negative effect.

X-Linked Disorders

• Most sex-linked disorders are X-linked.
• The Y chromosome contains:
  • SRY gene → determines testes formation and male sexual differentiation.
  • other genes in the male-specific Y region (MSY) → important for spermatogenesis.
• Mutations affecting the Y chromosome usually cause male infertility.
• Therefore, classic Y-linked Mendelian disorders are not generally transmitted.
• Most X-linked disorders are X-linked recessive.
• Heterozygous female carriers:
  • usually have one normal X and one mutant X.
  • each son has a 50% chance of receiving the mutant X and becoming affected.
• Female carriers usually do not show the full disease phenotype because they still have a normal allele.
• One X chromosome is randomly inactivated in each female cell, but enough cells usually retain an active normal X to prevent full disease expression.
• Affected male:
  • cannot transmit the disorder to his sons, because sons receive his Y chromosome.
  • transmits his affected X chromosome to all daughters → all daughters become carriers if the mother contributes a normal X.

KEY CONCEPT

• X-linked recessive → mainly affects males.
• Carrier mother → 50% of sons may be affected.
• Affected father → no affected sons through him.
• Affected father → all daughters receive his mutant X and become carriers.
• Female carriers are usually less affected because they also possess a normal X allele.

CONCEPTUAL EXAMPLES

• Carrier mother → one son receives mutant X → affected male.
• Carrier mother → another son receives normal X → unaffected male.
• Affected father + unaffected mother → sons receive father’s Y → sons unaffected through the father.
• The same affected father → every daughter receives his mutant X → all daughters are carriers.