Biotech exam 2

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Chromatin architecture serves as a control for which form of gene expression

transcriptional

How does chromatin architecture serve as the primary physical gatekeeper for gene editng machinery

1. High chromatin condensation decreases the specificity of engineered nucleases

2. Repair outcomes (Nhej vs HR) depend on the local nucleosome density and 3D genomic organization

What does 1 NUCLEOSOME consist of

1 nucleosome consists of a histone octamer with 146 Bp DNA tightly wrapped around it

What is the order of DNA packing

Nucleosome → chromatin → chromosomes

chromatin is many nucleosomes, while a chromosome is many chromatin

List the histones that make up the histone core and their functions

1. H1 - pulls the nucleosome together keeping it highly compact

2. H2A

3. H2B

4. H3

5. H4

H2A through H4 form chemical bonds with DNA to form compact nucleosomes

How many nucleotide pairs of DNA is associated with one nucleosome

146 Bp are wrapped around the core histones, while linker DNA strands bring the total Bp number to around 200

What charge interactions stabilize DNA and histone binding

negatively charged DNA creates tight bonds with the positive Lys and Arg groups on the surface of histone proteins. If DNA is tightly bound to the + charged histone residues, any genes present cannot be expressed

What is a histone

Histones are highly conserved, small, basic proteins

What is HISTONE acetylation

histone acetylation is a reversible modification of lysine residues in the N terminal of the core histones

this modification reduces binding to DNA and destabilized the chromatin structure, leaving DNA open for gene expression

Any mutated genes bound tightly to histones are NOT accessible for expression

How do histone proteins affect transcription

For transcription and/or replication to occur, chromatin must unwind to expose the template DNA needed for these processes to begin. Post translational modifications of histone tails determine chromatin condensation and therefore DNA accessibility

What is euchromatin

a not highly condensed region of chromatin, it is transcriptionally active

What is heterochromatin

a highly condensed region of chromatin that is transcriptionally silent

How does the + charge of lys interact with the - charge of DNA

Under normal cellular conditions, the + charge of lys interacts favorably with the - charge of DNA. The ACETYLATION of lysine will NEUTRALIZE the + charge of lys, and loosen the +/- binding, opening DNA for transcription/ RNA poly and TF binding

If a histone is acetylation, is transcription active or repressed

Active, because the + charge is neutralized and DNA can unwind from the histone core

If a histone is deacetylated, is transcription active or repressed

Repressed, as the + charge of lys interacts favorably with the - charge of DNA

What are the possible post translational modifications of the histone tails

1. Acetylation

2. Methylation

3. Phosphorylation

4. Ubiquitination

What enzyme transfers acetyl groups to Lysine to induce DNA transcription

histone acetyltransferases (HATS)

What enzyme removes acetyl groups from histones to repress DNA transcription

Histone Deacetylases (HDs)

What are the three proposed mechanisms of transcriptional repression mediated by cytosine methylation

1. Direct interference with transcription activator factor binding. TF factors cannot bind to highly methylated regions of DNA

2. Transcriptional repressors which bind to highly methylated regions, preventing TF and Poly 2 from binding

3. Inactive chromatin structure, where TF cannot access DNA

What is the significance of Heterochromatin in gene editing

Densely packed heterochromatin inhibits Cas9, ZFN, and TALEN binding and cleaving

What is the significance of Euchromatin in gene editing

Open chromatic regions are characterized by histone acetylation and DNA HYPOmethlation, these regions allow cas9, ZFN, and TALEN to easily bind to and cleave DNA

How does rotational and translational positioning affect Nuclease accessibility

DNA facing outward on the nucleosome surface may remain partially accessible to guide RNA search mechanisms, while DNA facing inwards toward the histone core is inaccessible

How does the local chromatin environment regulate how an introduced DSB is repaired

Heterochromatin favors NHEJ (which causes off site mutations), as the compact structure limits restriction enzyme access, delaying repairs and promoting error prone joining

Euchromatin favors HDR, as these regions are accessible to polymerase which promote precise template directed repair

How does nucleosome density regulate how introduced DSB are repaired

nucleosome positioning dictates the the exact boundaries of deletions, favoring INDELS

What is epigenome editing

The alteration of gene expression without making primary changed to the DNA. Instead of cutting or mutating the genetic code, epigenome editors add or remove chemical tags on DNA or histone proteins to turn genes on/off

What are the key differences between engineered nuclease (crispr, talens, zfn) vs an epigenome editor

An engineered nuclease introduced DSB or base changes which permanently alter their target DNA sequence through deletions, insertions, or rearrangements with a high risk of off target deletions or insertions. Epigenome editing on the otherhand modifies chromatin structure via methylation/acetylation, leaving the DNA sequence unchanged and a potentially reversable affect across cell divisions

What are the two core components of an epigenome editor

1. The targeting molecule (DNA binding protein) which retains its ability to bind to a specific target sequence guided by gRNA, but lacks endonuclease activity

Ex: dCas9, ZFPs, TALEs

2. Effector Module (an epigenetic modifier) which adds or removes acetyl/methyl groups from local chromatin

Ex: histone acetyltranferase to upregulate gene transcription OR a histone methyltransferase to silence transcription

Explain how Nucleases sensitivity to salt is useful to DNA analysis

To dissociate DNA from its histone core, nuclease will first digest any linker DNA in the sample, releasing just the nucleosome core particle. Then, high salt concentrations will break the octameric histone core into its subunits, releasing DNA into the solution.

High salt concentrations encourage DNA dissociation

Low salt concentrations encourage DNA binding

What is the significance of Micrococcal Nuclease (MNase)

MNase can induce DSB within nucleosome linker regions, but only single stranded nicks within the nucleosome itself. Because of this, Mnase can be used ot determine whether aDNA fragment of interest is within a nucleosome

Describe Ubiquitination

Ubiquitination involves one or more covalent additions of ubiquitin (protein) to the lysine residues of target proteins. This post translational modification is reversible, and is involved in the regulation of membrane trafficking and protein degradation

Describe the BRCA1-BARD1 tumor suppressor (protein) and its relation to DNA repair

The BRCA1-BARD1 tumor suppressor is an E3 ubiquitin ligase necessary for the repair of DSB by HR. Repression of these genes and their translated proteins prevents HR DNA repair and favors NHEJ/Cancerous mutations

What is Homologous recombination

HR is a biological process where nucleotide sequences are exchanged between two homologous molecules of DNA. It is known for its role in repairing broken DNA and shuffling genetic material

What are the three key steps of HR

1. Strand Exchange - occurs when a helper protein (Rad51) is used by an “invading” single strand to “attack” its matching DS DNA molecule (usually a sister chromatid)

2. D loop Formation - strand migration results in D loop formation, where an invading strand pairs with its complementary match on the sister chromatid. The cell then uses the sisterchromatid as a template to synthesize and rebuild the missing genetic sequence

3. Resolution - DNA strands are untangled and sealed, leaving a perfectly repaired DNA molecule

What is the significance of HR and genome editing

1. Error Free DNA repair - HR relies on an undamaged template to fix harmful DSB without mutation or loss of information

2. Driving Genetic Diversity - During meiosis HR cuts and swaps segments between maternal and paternal chromosomes, resulting in crossing over making sure that no two gametes are identical, providing genetic variation

3. Proper chromosome segregation

4. Guarding against Cancer - HR acts as a tumor-suppressing mechanism

5. Gene Editing - HR can be used for gene targeting by introducing custom DNA fragments flanked by sequences that match a specific target, this is used to trick a cells HR machinery into swapping out a target gene for a new one

Define Genome editing

a type of genetic engineering in which DNA is inserted, replaced, or removed using artificially engineered nucleases that create specific DSB and harness the cells mechanisms to repair the induced break by HR or NHEJ

What are the key differences between HR and NHEJ

HR is an error free mechanism that required an undamaged DNA template to fix a DSB. This is only active during S and G2, and is a slow complex process.

NHEJ is error prone and results in INDELS. No template is required, and it is a rapid response available throughout the entire cell cycle.

Describe the BRCA1 and BRCA2 pathway

1. Double stranded break occurs

2. BRCA 1 activates signals to pause the cell cycle

3. BRCA2 Loads strand exchange helper proteins (RAD51) to exposed ss DNA (step 1 of HR)

4. HR repair takes place as a flawless template directed fix

A mutation in BRCA1 or BRCA2 inhibits signaling pathways necessary for HR to take place

What occurs when a person inherits a mutation in BRCA1 and/or BRCA2

1. The HR pathway fails due to a lack of BRCA proteins/signaling

2. NHEJ is favored since HR cannot take place, leading to a build up of mutations and deletions of vital genes

3. These accumulated mutations result in genetic instability, accelerating the rate at which a cell can mutate, quickly transforming healthy tissue into aggressive cancers.

What is the result of chromosome duplication

Unbalanced gene dosage causing developmental abnormalities

What is the result of chromosome deletions

Possible total loss of gene functions

What are chromosomal fragile sites

GC rich regions of chromosomes are often poorly connected to the rest of the chromosome, putting the region at risk of a break and an extreeme loss of genetic material. A loss of this genetic material during development may result in decreased mental capacity

What are Zinc Finger Nucleases

ZFNs were the first programmable nuclease that can facilitate targeting editing of the genome through DSB at specific sites

What was the first human study involving ZFN

The first human ZFN study focused on mutating the CCR5 receptor located on T cells, inhibiting HIV from binding to and infecting T cells

What are the two domains of ZFN

1. The DNA binding domain (Zinc Finger Protein) - The left and right handed domains recognize DNA sequences on both sides of the helix, both domains must bind to the molecule for cleaving to occur

2. The DNA cleaving domain (Fok1) - Fok1 is a nonspecific nuclease, responsible for forming a dimer that cuts 7 bp of DNA at a time. This domain is nonspecific without the DNA binding domain

Describe the ZFN pathway and possible outcomes

1. The amount of donor sequence incorporated will decline with distance from the original break

2. NHEJ mutations at the cleavage site are more localized

What is the significance of having both the DNA binding domain and the DNA cleaving domain

When the DNA binding and DNA cleaving domains are combined, they make a highly specific PAIR of genomic scissors

What are the two outcomes of gene manipulations

1. Targeted Mutagenesis

2. Targeted gene replacements - replacement of an existing sequence with one designed in the lab

What organism is used to as a model for ZFN and why

Yeast is the most common model organism for ZFN, as its cellular machinery favors HR (mammalian cells favor NHEJ). The use of yeast allows for rapid phenotypic screening, and its DSB repair machinery is similar to mammalian systems, making findings directly applicable

Describe ZFN mediated genome editing

1. ZFN pair is delivered into the cell through transfection or electroporation

2. ZFN pair recognizes and dimerize around the target DNA site

3. If no repair template was added to the system, 1-20% of cells are repaired using NHEJ resulting in deletions

OR

4. if a repair template was added to the system, 1-20% of cells will be repaired using HR, resulting in gene integration

Define Transfection

Transfection is the process of introducing EXOFENOUS nucleic acids into eukaryotic cells to study gene expression. Cells may be stably transfected or transiently transfected

What is the core principle of transfection

to break through the cell membrane to successfully deliver exogenous material into the cell

Describe transient transfection

DNA is introduced into the cell, but is NOT integrated and instead remains as an episome (DNA in cytoplasm). Transient transfection shows only temporary expression, as the introduced DNA is lost as the cell divides.

Describe stable transfection

DNA is permanently integrated into the host cells chromosome, allowing for long term permanent gene expression that is passed down to daughter cells. This may be used for drug discovery or the generation or permanent cell lines

List the three types of transfection

1. Calcium phosphate transfection

2. Electroporation

3. Liposome mediated

What is calcium phosphate transfection

Transfection via the precipitation of DNA and Calcium Phosphate. This method may be used for stable or transient infection

What is electroporation

Exposure of cell membranes to high intensity electric pules, causing temporary destabilization of the cell membrane which allows for the entry of exogenous molecules. This however may cause cell death

What is liposome mediated transfection

The use of liposome forming lipids to introduce RNA, DNA, or CRISPR/cas9 components to a cell. This method is usually cell type specific

Define transDUCTION

Transduction is virus mediated transfer of exogenous material. Commonly used viruses are AAV and lentivirus

What three things must be considered when selecting transfection reagents and protocols

1. Desired cell type

2. Rare cell types such as neurons, which are more difficult to transfect

3. cytotoxicity parameters, with low cytotoxicity with high transfection being favored

What is the significance of ZFN

Double-stranded breaks are used to create site-specific mutagenesis that stimulates the cell’s natural DNA repair processes, such as HR and NHEJ. These processes are used to precisely target DNA sites, resulting in cell lines with targeted deletions, integrations, and modifications

What are the benefits of ZFN

1. Rapid disruption of, or integration into, genomic loci

2. mutations are permanent

3. work in a variety of somatic cell types

4. can create knock out or knock in cell lines in as little as two months

What are the drawbacks of ZFN

1. Edits occur in only 1-20% of the cell population (CRISPR is 90%)

2. High cost of ZF DNA binding domain construction

3. Off target mutations occur in 1/10 cells