Exam 4

CRISPR-Cas System (naturally occurring)

• Powerful tool used for editing genes

• CRISPR: “Clustered regularly interspaced short palindromic repeats”

• Found in prokaryotes

• used in immune system response → cuts DNA phages and plasmids to prevent damage to prokaryote

Cas9

Endonuclease that creates double-stranded breaks at specific sites ("CRISPR-associated proteins," which are enzymes that can be guided by CRISPR to cut DNA at specific locations.)

Single Guide RNA (sgRNA)

An engineered RNA that guides the CRISPR machinery to the specific location in the DNA that needs to be edited. Cas-9 then edits the DNA

Guiding region

20 nucleotide region that is complementary to the target region and defines the sequence that cas 9 cuts

Scaffold region

forms a multi-hairpin loop structure that binds in a crevice of Cas9 and stabilizes interaction between cas and guide RNA

Protospacer adjacent motif (PAM)

Immediately downstream of the target sequence.

• Short DNA sequence that signals Cas to bind to the DNA. When Cas9 binds, it separates the DNA strands of the adjacent sequence to allow binding of the sgRNA

CRISPR-Cas9 Steps

1. Recognition

2. Cleavage (sticky ends/bunt ends)

3. Repair (non-homologous end joining or homology directed repair)

Non-homologous end joining

• No requirement for a repair template or extensive DNA synthesis. Gluing pieces back together

  1. Broken ends recognized by heterodimer

  2. Ku acts as scaffold for recruitment of kinase, DNA ligase, and accessory factor (holds DNA ends together)

  3. Paired end complex then ligates compatible DNA ends together

Homology vs Non-homologous Directed Repair

Homologous → Repairs DNA breaks by using chromatids that are undamaged as a guide.

Non-Homologous → Rejoins DNA strands at the site of a break with no template. Quicker, but error-prone → mutations (basically gluing together).

CRISPR applications

• Livestock pests

• biomedical models

• productive traits

• disease resistance

• animal welfare

Maybe?

Genetic Plasticity

Sex determination in alligators and sea turtles depends on temperature of eggs at fertilization

Fetal Programming

Events involved in normal fetal
development have long-term effects
and influence health during adult life

Epigenetic effect of social stress in fish

Can turn female-to-male sex change in bluehead wrasse.

What allows for this? A cell that is able to differentiate into hundreds of different cell types.

What is Epigenetics?

Genes that express themselves differently, although the underlying DNA sequence is the same (same car materials but different overall car)

How does epigenetics work?

• Cell type identify: inherited from one generation of cells to the next

• Dynamic nature: constantly changing in response to the environment

• Reversible

DNA methylation

A methyltransferase enzyme adds methyl groups (CH3) to DNA, altering gene expression.

→ methylation of promoters seems to prevent binding of transcriptional factors to the promoter thus shutting down expression of the gene/allele

What is the purpose of DNA methylation?

• Acts to repress/activate gene transcription

• Needed for normal development

• Involved in genomic imprinting

• X-chromosome activation

• Repression of transposable elements, aging, carcinogenesis

Histone Modifications

Act to package DNA into chromosomes.

• is a post-translational modification (PTM) to histone proteins: methylation, phosphorylation, acetylation

• PTMs can impact gene expression

What does examining histone modifications at a particular region reveal?

Reveals gene activation states, locations of promoters, enhancers, and other gene regulatory elements

Histone Acetylation/Deacetylation

Acetylation → adds acetyl group to histones. Relaxes chromatin to make it easier to access DNA and promote gene expression

Deacytlation → Removes acetyl groups to condense chromatin. Inhibits gene expression by making it harder to access DNA

Acetylation and gene regulation

Acetylation adds (-) charge to lysine on histone tails that extend outward of nucleosome → (-) repels (-) DNA → relaxed chromatin structure → transcription factor binding/increase in gene expression

Cat colors

• O gene located on X chrom. Orange = X^B and black = X^b.

• Orange is dominant

  • Females with XBXb will be mixture of orange and black.

X chromosome inactivation

• Epigenetic mechanism controlled by DNA methylation

• 1 X-chrom is inactivated in females → high levels of DNA methylation, low levels of histone acetylation, other marks ass. with gene silencing

• All cells that descended from original will have X-inactivation

Random X inactivation

Genomic Imprinting (imprinted genes)

• expressed from only one of their two alleles, depending on parental origin

• Some are maternally expressed, some paternally expressed

• Essential for development/growth of embryos/tissues of placental animals

• Occurs in clusters of genes on chromosomes

• Genes inherited by one of the parents can affect fetal growth/hormones/other elements based on if they are active from parent or not.

Modes of inheritance

Autosomal dominant/recessive, X-linked dom/rec., mitochondrial

Genomic imprinting phenomena in livestock

• Associated with AI repro and cloning

• Large offspring syndrome

• natural genetic variation important for production traits and values

• Callipyge phenotype in sheep

Callipyge

• Overly muscular rear legs with reduced fat content

• Decreased muscle proteolysis (breakdown of proteins into amino acids) caused by elevated calpastatin levels

• Heterozygous for mutation, polar overdominance, CLPG locus

• (polar overdominance = offspring shows superior traits compared to parents)

Large Offspring Syndrome (LOS)

Cattle sheep, etc. → Parturition difficulty, organ defects in offspring, neonatal losses.

• Triggered by in-vitro fertilization

• Significantly increased birthweight

• Intrinsic to fetus rather than maternal malfunction

Why are imprinted genes considered candidates causing LOS?

• Embryos show higher sensitivity to external stimuli during in vitro production

• Imprints are established prior to implantation

• Many imprinted genes control growth and early embryonic development

IGF2 candidate for LOS

Humans: Improper methylation of IGF2 locus → BWS and Angelman syndrome

Sheep: complete loss of methylation in IGF2

Paternal Conflict Theory

• Fathers genes gain greater fitness through offspring success (at expense of mother)

• Mother needs to provide sufficient resources

• Paternal = growth promoting genes, Maternal = growth-limiting

DNMTs

• DNMT1 : maintains DNA methylation after each replication by using parental DNA strand as template

• DNMT3a/DNMT3b: establish new DNA meth. patterns to unmethylated DNA

• DNMT3L: cant meth., reacts with DNMT3a to form dimer that ensures DNMT3a methylation activity

Agouti Mouse

Supplementation of methyl donors to maternal diet during pregnancy affected coat color and obesity

The dutch famine

The timing of the insult during pregnancy determines phenotypic outcomes of the offspring.

→ Early gestation: diabetes, obesity, cardiovascular disease

→Mid-to-late: significantly reduced birth weight

When children grew up/had children, children also smaller

Nutrient restriction can permanently impact adult health via epigenetic mods.

Effects of Chinese Famine

Risk of hypertension was not elevated in those exposed to famine during fetal development only. Higher in those exposed during infancy and increased short stature. Did not increase risk of obesity.

Queen Bee Making

• Royal jelly diet during larval development

• Royalactin induces differentiation

• Increases body size and ovary development/shortened development time

• Differences in methylation patterns, clustered in areas of genes where splicing occurs

• Meth. of CpG sites in the CpG islands of promoters is correlated with gene expression

Dads effect?

Small swedish community: food availability for paternal grandfather effected the lifespan of his grandchildren

Shortage associated with extended lifespan of grandchildren.

Mother Diet Hypothesis

Different maternal diets during pregnancy induce gene expression changes and DNA modifications in fetal muscle and adipose tissues in sheep

miRNAs: an epigenetic mechanism

The study of changes in gene expression without alteration of genetic code itself

miRNAs are…

• Indicators of embryo quality and development

• Regulators of genes important to pregnancy

• A mode of communication to the mother at early pregnancy

• Signals which travel to distant bodily tissues and affect gene expression

• important biomarkers

How do miRNAs regulate genes?

Translational block (partial match with mRNA) vs. mRNA degradation (perfect match)

RISC

Where can miRNAs be found?

Reproductive tracts, gametes, zygotes, and embryo development

How to test fetal/mother signaling

• Supplement miRNAs secreted by the embryo to maternal cells in culture

• Test whether maternal cells uptake embryonic miRNAs

Exosomes:

Extracellular vesicles which can transport functional miRNAs and mRNAs throughout the body

Advantages of using miRNAs as biomarkers

Not invasive for early diagnosis, highly stable, highly reproducible, consistent across individuals, expressed across species, offers panel of markers for better diagnosis of condition

Small molecules with large influences

• travel in circulation

• affect gene expression in other cells

• multiple mRNA targets

• multiple miRNAs

• strong influences over pathway regulation

What do sperm contribute (PATERNAL CONTRIBUTION)

Methylation patterns, mRNAs, small non-coding RNAs, and proteins

Bull Fertility Hypothesis, Results

Bulls of different fertility will have different DNA methylation signatures that affect preimplantation embryo.

→ Development/morphology of embryo same between high/low

→ Higher methylation in high.

Differently expressed genes?

If an observed difference/change in read counts or expression level between two conditions is statistically significant

DMRs vs DMCs

DMRs → Differentially methylated regions (sum of methylation of groups of cytosines)

DMCs → Differentially methylated cytosines (only one CpG)

Sheep Study

Methylamine fed to sheep → affected everywhere in genome → methyl marks influenced by the paternal diet may be transmitted to the embryo and affect the transcription efficiency

Transgenerational study

• Needs to be seen in every generation to be transgenerational

• Scrotal circumference significant → correlated with fertility and indirect teste function → weight significant in females

Bull Fertility < Female Fertility

Resumption of cyclicity postpartum, calving interval, submission rate (% of herd that received 1+ insemination within # of days) > significant variation of sperm (can check with motility, morphology, and CASA but don’t measure transport through repro tract or ability of sperm to travel).

Conceptus Growth

Spherical → tubular → filamentous (mare remains spherical) → unique to ruminants

Maternal Recognition

• Day 16-17

• Ovulation → formation of CL → secretes progesterone

• Not pregnant/not recognized → PGF2a is secreted → kills CL

• Conceptus secretes interferon tau → sensed by endometrium → blocks endo. from secreting PGF2a

Embryo fertility Study 1 vs study 2

Study 1 → Those with high fertility sperm will more likely develop an embryo than those with low

Study 2 → Conceptus recovery high in HF than LF

Why day 7 and 15? → key checkpoints, despite no difference in length of recovered conceptuses → those that do survive are comparable between the two

Accessory Sperm

Population of sperm able to get through female repro tract and partially penetrate zona pellucida

Embryonic Genome Activation

• Up until the 8-16 cell stage, the embryo depends on maternal RNAs for survival. EGA marks the transition from maternal resources to own genetic instructions for development.

Differently Expressed Genes

A gene is declared differentially expressed if an observed difference, change in read counts, or expression levels between two experimental conditions is statistically significant.

Genomic Imprinting vs Monoallelic Expression

• Monoallelic: independent of the parent of origin, One gene, one allele, randomly expressed. Involved in immune response/Neuronal development (mainly DNA sequence polymorphisms)

• Genomic: Parent-specific gene expression, controlled by DNA marks (to make active or not), important for fetal development/growth.

Embryo Splitting

An early stage embryo is manually divided into two individual cells that then grow into two identical embryos.

Somatic Cell nuclear transfer

The nucleus from the somatic cell is transferred into an oocyte that has had its nucleus removed.

Reprogramming and epigenetics

Reprogramming involves altering the characteristics of a cell, often by manipulating its epigenetic state.

Epigenetics involves the mechanisms underlying changes in gene expression and cellular phenotype.

Applications for transgenic animals (genetically modified)

• Disease resistance

• To make therapeutic proteins in eggs.

• Make antibodies for humans in cows (transchromosomal)

• To reduce phosphorus in manure in pigs

• To produce milk with longer shelf life

Genetic Engineering Concerns

Introduces genetic change to animals by putting modified genes in newly fertilized eggs.

• Is it safe? Unnatural?

• Animal welfare

• Environmental concerns

• Zoological disease possibilities?

• Animals escaping and breeding with wild animals = ruin natural resistance

• Larger loss of embryos, neonatal, etc.

Non-Coding RNAs

Type of RNA molecule that does not code for proteins. Instead, it performs various regulatory functions within the cell.

Imprinting Enhancers vs Promoters

Enhancers →short DNA sequences that regulate gene expression by binding to transcription factors

Promoters → Serve as binding sites for RNA polymerase and transcription factors

Steps in Genomic Selection

1. Build a reference population

2. Estimate SNP effects → data analysis

3. Genomic Selection → use marker information (young animals as selection candidates)

4. Make selection → superior animals with high gEBV

Genomic Selection Applications

• Predict hard-to-measure traits, low-heritable traits, and sex-limited traits

• Disease, meat and carcass traits

• Accelerate genetic progress

• Potential application for treatment of livestock/companion animals