MCB104, Lecture 16 - Genome Editing

If full genome screen, is it forward or reverse?

Forward Screen, I have no clue what’s going on and I need to make an hypothesis, I am testing everything.

If I apply a filter during testing to a genome sequence (Full to ANYTHING lowered) , is it forward or reverse?

Reverse Screen.

What is a transgenic organism?

• Contains a transgene from another individual of the same species or a different species

• For transgene to be propagated between generations in a multicellular organism transgene must be in cells that become gametes

What is a Transgene?

• A gene derived from another species/individual

• Can be made in vitro using recombinant DNA technology

• Introduced into eukaryotic cells by chemical treatment (yeast) or injection

What is DNA Delivery Methods: CHEMICAL

• HEAT SHOCK + (CALCIUM CHLORIDE)

  • Standard methods for transformations

  • Apply solution of calcium chloride and DNA to cells, stress cells with pulse of heat

  • Combination of CaCl₂ and heat opens pores allowing DNA uptake

• LIPOSOMES/POLYMERS

  • Use positively charged molecules to envelop DNA

  • Forms complexes the cells can take up

• NANOPARTICLES

  • Bind DNA to nano particles that cells will take up via endo or pinocytosis

What is a DNA Delivery Methods: PHYSICAL

• Pore forming methods

  • Pore/holes are opened in membrane via application of energy

    • Electrical, ultrasonic, light, etc

• Microinjection

  • DNA is injected indirectly into either the cytoplasm or ideally the nucleus, bypassing membrane barrier

• Biolistics

  • A “Gene Gun” that shoots microparticles coated in DNA into target cells

  • Usually for plants

  • OG is a modified BB gun

What is DNA Delivery Methods: VIRAL/MICROBIAL

• Viral

  • Create a functional virus but with an infection deficient genome + desired DNA sequence

  • Expose cells to virus

  • Virus introduces DNA to cell

• Bacteria

  • Agrobacterium infects plants and fungi and forms gails (tumor like growths)

  • Has special T-DNA vectors (plasmids) that are transferred to neighboring plant cells and contain growth factors

  • Alter vectors to contain desired DNA and selective marker (drug resistance)

  • Infect plants with modified agrobacterium, desired DNA is inserted into plant

What are some DNA Integration Methods: RANDOM?

• Random

  • Occasionally DNA will randomly integrate into the genome

  • This can be facilitated by inducing double strand breaks

What are some DNA Integration Methods: TRANSPOSON?

• Transposable elements naturally will insert themselves into genomes at random sites

• Design transposon that contains only the sequences required for insertion and the desired sequence

• Apply to cells along with transposase to insert sequence into genome

What are some DNA Integration Methods: HOMOLOGOUS RECOMBINATION?

• Flanking desired DNA with sequences homologous to target sequence causes DNA repair via homologous recombination, inserting DNA in targeted location

What are Pronuclear Injection Method (Transgenic Mice)?

• Fertilized eggs harvested from female mouse

• Transgene is injected into one of the pronuclei of the fertilized egg

• 25-50% of the time the injected transgene integrates into the mouse genome in a random location

• Mice injected as embryo born

• If transgene integrated after cell division, the transgene will be in some cells and not others (mosaic)

• Mice are bred to produce stable transgenic lines

What are Transposon Method (Transgenic Flies)?

• P element is a type of transposable element

• A transformation plasmid is constructed to contain the P element inverted repeats surrounding a transgene and a visible marker

• A helper plasmid contains the transposable gene

• The transformation plasmid and helper plasmid are injected into fly embryos

• Resulting adult flies are mosaic

• Visible marker helps identify transgenic flies in subsequent crosses

What are Agrobacterium Method (Transgenic Plants)?

• Like a DNA transposon, the Ti (tumor inducing) plasmid contains left and right border sequences (LB and RB) surrounding vir genes

• The transferred DNA is integrated into the plant genome through the action of trans-acting Vir enzymes

• Recombinant T-DNA plasmid is constructed

• T-DNA plasmid and helper plasmid are sprayed on plants

• DNA is transferred and integrated into plant cell genome

• Individual transgenic plant cells are selected by resistance to herbicide and grown into plant

What are some uses of transgenic organisms?

• Identifying causative genes

• Creating reporter genes (transcriptional and translational)

• Studying protein function

• Producing pharmaceutical/industrial proteins/compounds

• Improving agriculturally relevant organisms

Given a recessive mutant that results in defective eyes, how can we identify the causative gene?

• Use mapping to identify approximate location

• Construct transgene expressing genes in that region

• If a transgene rescues the mutant phenotype, i.e. reverts to wildtype, that is most likely the causative gene

What is Reporter Constructs?

• Any protein that can be used to indicate protein abundance

• Examples:

  • lacZ

  • FLuorescent Tags: GFP, RFP, mCherry, etc

  • Luciferase

• LacZ and Luciferase rely on chemical reactions allowing them to be used to calculate amount of functional enzyme present

  • Luciferase doesn’t produce light but catalyzes the reaction to produce light

How do Transcriptional Reporters work?

• STEPS

  • Clone the regulatory sequences surrounding a gene

  • Replace the protein coding region with a marker protein (GFP)

  • Insert transgene into the organism at a random location

  • GFP expression will correlate with mRNA expression of the original gene

• SOME USES

  • Determining expression pattern of a specific gene

    • Includes cell type, timing, and induction/repression

    • Identifying regulatory proteins and sequences

    • Monitoring certain biological responses

      • E.g.: Immune reporter that activates upon infection

  • Nomenclature: Pname::GFP

What are Translational Reporters

• STEPS

  • Clone the gene and its regulatory sequences

  • Insert the marker protein sequence in frame with the protein

    • Usually at the C terminus

  • Confirm new fusion proteins retain its function while being tagged

• SOME USES:

  • Observe localization protein

  • Directly measure levels of target protein

  • Epitope tagging for antibody-based methods

• Nomenclature: name::GFP

What are Protein Factories?

• Some human proteins used as drugs can be produced in bacteria, but not all, due to posttranslational modifications

• Transgenic mammalian cells in liquid culture used to make pharmaceutical proteins

  • Factor VIII protein: blood clotting factor

  • EPO: stimulates red blood cell production

• Plant cells used to make glucocerebrosidase for people with Gaucher’s disease

Describe an example of protein factory

• Transgenic mammals produced by pronuclear injection

• Expression directed to mammary glands and gene product is secreted in milk

• Identical clones of high producing animals can be made using reproductive cloning

• In this case goat milk and antithrombin III which prevents improper blood clotting

How do we investigate function after identifying a gene of interest?

• Can introduce edited version to investgate roles of different parts of protein

• By deleting specific parts of protein, you can determine function

What does TOE-2 with their cell cortex interacting ___ and ____ domains?

DEP and 3MAPK

What does deleting the MAPK interacting domains to TOE-2 do?

Membrane localization

What does deleting the DEP interacting domains to TOE-2 do?

Apoptosis defects

What are Transgenic Disease Models

• Generate an animal with an mutation that corresponds to a human disease allele and a similar disease phenotype

• Transgene added to normal organisms, so only dominant mutations can be modeled

• Mice are common model organism. Primates may be better models for complex neurological disease

  • First primate model was for Huntington disease (Rhesus macaque)

  • Future of primate models unclear (ethical issues)

What are Genetically Modified Organisms

• Many organisms have been gene edited for agricultural purposes

• Some example modifications

  • Herbicide resistance

  • Pesticide production

  • Increased growth/yield

  • Decreased height (same yield)

  • Improved drought tolerance

  • Improved disease resistance

• GMOs allow for more efficient production of more food in less space, reducing environmental impact of farming

What are some examples of GMO Examples

• More than 100 different transgenic plant species have been created

  • ROUNDUP Soybeans are herbicide resistant (over 90% of US soybeans)

  • Corn expressing Bt protein protects the plant from corn-borer moth caterpillars

  • In 2015, first GM animal: GM salmon that expresses growth hormone - approved for human consumption

What are some GMO concerns

• Few scientists now believe that eating food from GM organisms poses a direct danger to humans

• Human/economic concerns

  • Few large agricultural companies have consolidated power

  • Farming communities may be disrupted

• Scientific concerns

  • Potential environmental consequences, such as transfer of transgenes to wild organisms

What are Transgene Limitations?

• Transgene regulation depends on including all relevant regulatory sequences

  • If you’re making transcriptional reporter and miss an LCR 50kb upstream, your reporter will NOT be accurate

• Many integration methods insert multiple copies of a given transgene, causing possible dosage problems

  • Most often a problem for translational reporters

  • Sometimes a problem for transcriptional by titrating transcription factors

• If you’re trying to test a mutant allele, the endogenous gene is still present

  • Must independently knock out endogenous gene sequence as well as reintroduce it as a transgene

What is Targeted Mutagenesis?

• Targeted mutagenesis enables scientists to change specific genes in virtually any way desired

• Process called gene targeting

  • Specific gene mutagenized in vitro

  • Mutant DNA put into cells

  • Rare homologous recombination replaces normal gene with mutant gene

What is Gene Knockout?

• Gene Knockout: Functional allele of a specific gene replaced with an amorphic (nonfunctional) allele

  • Amorphic allele often constructed by inserting selection marker

What is Gene Knockin?

• Gene Knockin: New sequence is added to specific gene

  • Reporter, epitope tag, functional domain, mutation, etc

  • Generally most accurate translational reporters are knockins

What is the difference between Gene Knockout/Knockin and transgenes?

• Distinction from transgenes: Edits occur at endogenous locus

  • Transgenes integrated elsewhere

What are Knock Out Mice Steps?

• Generate construct with neo^r gene disrupting target gene

• Isolate ES cells from agouti mice

• Treat cells with construct

• Select for integration with neomycin

  • Only cells where homologous recombination inserted the construct will survive

• Inject the neo^r cells into blastocyst from black mice

• Implant blastocyst in another female mouse

• Progeny will be chimeras

  • Composed of cells from different individuals

• Chimeras are crossed with black mice.If the edited cells are in the germline this will produce agouti progeny

  • Agouti allele is dominant to black

• Cross agouti progeny to make homozygous knockout mice

What are Conditional Knockout (Mice)

• Knockout of essential genes are uninformative

• Conditional Knockouts only eliminate gene function under specific circumstances

• Common examples:

  • Environmental signal (ex: heat shock)

  • Cell type specific (Eyes only)

What is the Cre/LoxP Recombination System?

• Bacteriophage Cre protein causes crossing-over between 2 loxP DNA sequences

What are Floxed Genes

• Floxed; Flanked by LoxP

• For essential gene knockouts

  • Generate construct with LoxP sites flanking an exon and neo^r

  • Insert into ES cells as before

  • Select for neo^r as before

  • Transiently Induce Cre expression

    • Often via heat shock promoter trasngene

  • Identify cells with no neo^r and functional gene

  • Make chimeras with those cells, perform crosses to make homozygous for Floxed gene

What are Conditional Knockouts?

• Perform crosses with floxed mice to mice with Cre transgene under a conditional regulatory sequence

• In mice with both floxed gene and conditonal Cre, cells expressing Cre will knockout the floxed gene

  • In example on right: eye specific Cre transgene

What are Knockins (With Cre/LoxP)

• Knockins alter instead of deleting

• Can use same methods as before except the construct has

  • An edited sequence in gene

  • Floxed selectable marker in an intron near the site you want to edit

• Inducing Cre expression removes the selectable marker, resutling in the only edit being the desired one

• Can be used for disease models by knocking in human disease alleles into mice

  • FGFR3 disease allele knockin shown on right

What is CRISPR/Cas9 Gene Editting

• Several newly developed gene editing technologies allow alteration of the genome of virtually any organism

• CRISPR: clustered regularly interspaced short palindromic repeats

  • Serves as antiviral immune system in bacteria

• Cas Proteins: CRISPR associated proteins; endonucleases

• CRISPR/Cas9 System has an sgRNA that is complementary to target site of interest

• In nucleus, Cas9/sgRNA seek out designated genomic sequence, make double strand break in target DNA

What is CRISPR/Cas9 in Bacteria - COMPONENETS?

• 3 Components

  • Cas9: The Endonuclease

  • tranrRNA: Links the Cas9 protein ot the crRNAs

  • CRISPR: Creates the Pre-crRNAs consisting of repeats and spacers

    • Spacers are fragments of previous viral insertions

What is CRISPR/Cas9 in Bacteria - PROCESSING ?

• Processing

  • Cas9 binds tracrRNA

  • Pre-crRNAs are cleaved into mature crRNAs, with guidance from tracrRNA+Cas9

  • crRNAs are bound to the Cas9+tracrRNA complex via pairing with the tracerRNA

  • Recognition of DNA with complementary sequence targets genomic region

  • PAM site allows cleavage

What are PAM sites?

• Cas9 will only cleave DNA if the 20 bp of genomic DNA complementary to the sgRNA are followed by a triplet of DNA called the Protospacer Adjacent Motif (PAM) site: 5’NGG

  • sgRNA mustbe designated to target parts of the genome with a correctly oriented PAM site

  • Other Cas9 related proteins have different PAM sites, expanding options

What is CRISPR/Cas9 mediated mutagenesis

• Instead of tracer and crRNA, single combined sgRNA used instead

• 2 options for mutations

  • Allow double stranded break to be repaired by nonhomologous end joining, can result in small indels

  • Introduce a DNA molecule with the desired mutation, and double stranded break repair by homologous recombination will form a knockin

• EASIER TO KNOCKOUT THAN KNOCKIN

What are CRISPR Variants: CRISPR Interference?

• Uses catalytically dead Cas9 (dCas9) with no endonuclease activity

• Sterically blocks transcription by occupying promoter or exon

• In eukaryotes, can be connected to a repressor domain for stronger repression

What are CRISPR Variants: CRISPR Activation?

• Uses dCas9 fused to transcriptional activation domains

• Effectively a transcription factor with the sgRNA as the DNA binding domain

What are CRISPR Variants: CRISPR Base-editors?

• Fuse dCas9 or nicakse Cas9 (nCas9) with base editors that induce specific mutations at the targeted site

  • Example: Deminases that can cause transition mutations

What are CRISPR-based screen?

• Can use CRISPR library to screen many genes at once

• Generate gRNAs and insert into vectors

• Apply mix of vectors to cells

• Screen cells for hits

• Sequence hits to identify gRNA used

What are CRISPR/Cas9 Key points?

• Flexible targeting system, but 22bp of recognition is too short for perfect specificity which means off target effects are common

• Requires both Cas9 and sgRNA: 2 methods

  • IN VITRO Preparation: Purified Cas9 and synthesized sgRNA are incubated together and injected into the target

  • IN VIVO Expression: Cas9 and/or sgRNA are expressed from transgene in the organism and assemble in the cell

• Functions fairly well in most systems with some adaptation

• Induction of DSBs at targeted sites significantly boosts homologous recombination at those sites

What is Gene Therapy?

• Gene Therapy: Introducing a therapeutic gene into the somatic cells of patients

• Different strategies are required for different diseases

  • Disease caused by loss-of-function: add wild-type copy of gene

  • Disease caused by gain-of-function: therapeutic gene must inactivate disease gene or protein product

  • Diseases are complex origin: target gene for a genetic pathway involved

What are some gene therapy methods: IN VIVO

• Therapy delivered to somatic cells in the body

• EX: Injected into retinal cells or inhaled into lungs

What are some gene therapy methods: EX VIVO

• Cells removed from body, treated, and then put back in

• EX: Bone marrow cells

What are some Viral Vectors for Gene Therapy?

• 2 types of viruses are common: retroviral vectors and adeno-associated viral vectors (AAV)

• Recombinant retroviral genome

  • Has therapeutric gene and genes for packaging virus particles

  • Doesn’t have most of viral genes

How do we package viral vectors?

• In order to prevent the viruses from replicating, the genome need to be truncated

• However, that would prevent virus assembly

• Solution: Special Packaging cells with viral genes without packaging sequences

• Virus particles form, but only DNA packaged is the desired sequence

What is Retroviral vs. AAV vector Gene Therapy

• Retroviruses

  • Integrate into genome

  • Can cause mutations

• AAV Vector

  • Doesn’t integrate into genome

  • Therapeutic vector eventually degrades

What are example application of Retroviral gene therapy?

• Treated with retroviral gene therapy using ex vivo modification of bone marrow cells

• Patients regained immune system function

• Mutation resulted in 4 children with leukemia

What are example application of AAV Gene Therapy?

• Recombinant AAV vectors injected into retinal cells

• Patients regained sight

• Patients may require additional treatments when vector degrades

What is CRISPR/Cas9 Gene therapy?

• Induction of DSBs at targeted sites significantly boosts homologous recombination at those sites

• Efficiency is high enough to permit knockin/knockout without some of intervening steps previously required

How do we repair sickle cell disease; HOMOLOGOUS RECOMBINATION STEPS

• Red blood cell precursors removed from person with sickle cell disease

• Expose to Cas9, sgRNA against HbB^S, and DNA with wild type B-globin allele

• Cas9 cuts sickle cell allele, homologous repair converts it to wild type

How do we repair sickle cell disease; FETAL HEMOGLOBIN ENHANCER KNOCKOUT

• Red blood cell precursors removed from person with sickle cell disease

• Expose to Cas9 and sgRNA against fetal hemoglobin regulatory enhancer

• Cas9 cleaves enhancer sequence until indel inactivates it

• Loss of enhancer reactivates HbF

• Knockouts are easier than knockins