Lecture 18 Genetic Engineering: GMOs, Genome Editing, Gene Therapy and Synthetic Biology

genetic engineering

defined as the process of using recombinant DNA technology to alter the genetic makeup of an organism

transgenics

GMOs are also known as ___ because a gene or genes are transferred from one or more species into another species

plasmid

Many species of bacteria have extra chromosomal DNA known as ___.

cloning vector 

This plasmid can be extracted and used as a __

restriction enzymes

The segment of DNA containing the gene of interest is isolated by cutting the segment using ___. This gene of interest comes from another species 

plasmid vector

The __ is also cut with the same restriction enzymes used in cutting this DNA segment containing the gene of interest

recombinant plasmid or recombinant DNA

The plasmid carrying the gene from another species is known as ____ hence the term recombinant DNA technology

bacterial cell

This recombinant DNA is inserted into a ___ and grown in culture producing clone of cells containing the cloned gene 

expressed

Then the inserted gene is __ which means that it gets transcribed and translated.

steps in involved in producing GMos?

• GMOs are also known as transgenics because a gene or genes are transferred from one or more species into another species

• Many species of bacteria have extra chromosomal DNA known as plasmid. This plasmid can be extracted and used as a cloning vector 

• The segment of DNA containing the gene of interest is isolated by cutting the segment using restriction enzymes. This gene of interest comes from another species 

• The plasmid vector is alco cut with the same restriction enzymes used in cutting this DNA segment containing the gene of interest 

• Then the gene of interest is inserted into the plasmid. 

• The plasmid carrying the gene from another species is known as recombinant plasmid or recombinant DNA hence the term recombinant DNA technology 

• This recombinant DNA is inserted into a bacterial cell and grown in culture producing clone of cells containing the cloned gene 

• Then the inserted gene is expressed which means that it gets transcribed and translated.

Gene of interest could be used to:

• alter bacteria for cleaning up toxic waste

• gene that codes for growth hormone to treat stunted growth

• gene that codes for protein that dissolves blood clots in heart attack therapy 

bacterium

The gene of interest could be from a ___ and inserted into plant cells to produce pest resistant plants for example: Bt corn, Bt eggplant or Bt cotton

Genetically modified plants

Bt corn, Bt eggplant or Bt cotton

It stands fior BACILLUS THURINGIENSIS

What does Bt stand for?

BACILLUS THURINGIENSIS

__ produce crystal proteins or proteinaceous inclusions which are also called delta endotoxins that are toxic to certain pests 

agrobacterium tumefaciens

The DNA segment containing genes that code for these endotoxins are cut with restriction enzymes and inserted into a plasmid vector like the Ti or tumor inducing plasmid obtained from the bacterium ___ which has now been made as taxonomic synonym Rhizobium radiobacter

micropropagation or tissue culture 

The recombinant plasmid is inserted into plant cells generated via __

immature seed embryo 

An alternative is to directly insert the Bt gene into plant tissue such as an ___

seed embryo

If a ___ is used this would result in the Bt gene being introduced to some but not all of the cells in a young plant 

beta galactosidase

The lacZ gene codes for the enzyme __

digestion of lactose

The lacZ gene codes for the enzyme beta galactosidase which when expressed will catalyze the ___ present in the culture medium

chromogenic substrate

The culture medium contains a __ so that when a bacterial colony shows a beta galactosidase activity the colony will turn blue

disrupts

DNA inserted into the Polylinker region __ the lacZ gene resulting in white colonies that allow direct identification of bacterial colonies carrying cloned DNA inserts 

Polylinker region

The __ contains the restriction enzyme cutting sites

selectable marker

The plasmid also contains a __, the gene for resistance to the antibiotic ampicillin  

antibiotic ampicillin

Only bacteria that have taken up the plasmid containing this antibiotic resistance gene will grow in a medium which contains the __

RESTRICTION ENZYMES

__ are endonucleases which means they cut or digest within the nucleic acids 

• These enzymes are derived from bacteria

• These enzymes cut DNA at specific sites

EcoRI (EcoR1)

The restriction enzyme known as __ is derived from the bacterium Escherichia coli

• The recognition sequence of this enzyme is G A A T C

between G and A

When it sees the G A A T C sequence in a DNA, EcorI it cuts the DNA __ on either strand and producing fragments with sticky ends

PALINDROMIC SEQUENCE

A ___is a sequence made up of nucleotides in a double stranded DNA or RNA molecule wherein reading in a certain direction say 5 prime (5’) to 3 prime (3’) on one strand matches the sequence reading in the same direction that is 5 prime to 3 prime on the complementary strand  

Alul and HaeIII (Alu1 and Hae3)

Some restriction enzymes produce fragments that have sticky ends, others produce fragments with blunt ends like __

true

recognition sequences are all palindromic (t or f)

methods of delivering foreign DNA into cells

• gene gun

• electroporation

gene gun

device that can be used to transfect cells with foreign DNA by bombarding the target cells with micro particles of heavy metals that are coated with DNA 

electroporation

electric pulse is used to transfect cells with foreign DNA 

Golden Rice 2 

GMO crop approved by the US Food and Drug Administration in 2018

Golden Rice 2 genes

• psy gene

• crt1

psy

__ gene from maize or corn which codes for the enzyme phytoene synthase

crt1

__ from the soil-dwelling bacterium Erwinia uredovora 

Phytoene synthase

is involved in the biosynthesis of carotenoids.

• It catalyzes the conversion of geranylgeranyl pyrophosphate to phytoene.

Phytoene desaturates

__ are involved in carotenoid biosynthesis.

• They catalyze the conversion of phytoene into lycopene.

rice plants have endogenous enzymes that process the lycopene to beta carotene in the endosperm giving golden rice to its distinctive yellow color 

What gives golden rice yellow color?

vitamin A

Beta carotene is a precursor of __

23 times

Golden rice produces up to __ as much beta carotene as the original golden rice

luciferous gene

a tobacco plant carrying __ from firefly so that the plant can light up in the dark,

green fluorescent protein

 zebrafish carrying the gene for __ from jellyfish  

transgenic sheep and transgenic goat

There have been attempts to produce pharmaceutical products using transgenic animals like __

goat oocyte

The human gene for growth hormone can be microinjected into a __ and then the oocyte is fertilized and allowed to develop and then implanted in a surrogate mother 

milk of transgenic goat

The human growth gene is expressed in the __

reproductive cloning

Differentiated cells of this transgenic goat can be used for __ to produce more goats that carry the human growth hormone gene

ANTITHROMBIN

a circulating plasma protein that plays an important role as regulator of blood coagulation by inactivating several enzymes of the blood coagulation or clotting cascade

• first therapeutic protein produced in the milk of transgenic animals specifically in transgenic goats to be approved for marketing

genome editing

It is also called the genome engineering or gene editing

• It is a genetic engineering technique in which DNA segment is inserted, deleted, modified, or replaced at specific or targeted sites in the genome of a living organism

eugenics

is a set of beliefs and practices that are aimed at improving the genetic quality of a human populationE

clustered regularly interspaced short palindromic repeats - CRISPR associated systems

CRISPR-Cas stands for?

• prokaryotic immune system that confers resistance to foreign genetic elements such as those found in plasmids and viruses 

• This system provides a form of acquired or adaptive immunity to the prokaryotes 

What is the function of CRISPR-Cas?

CRISPR RNAs or crRNA

__, an operon of cas genes encoding the cas protein components

transactivating crRNA or Tracer RNA

__ is a small RNA that is trans encoded upstream of the CRISPR-Cas locus

• was reported to be essential for crRNA maturation

novel repeat-spacer unit

After insertion of exogenous DNA from viruses or plasmids, a cas complex recognizes foreign DNA, cuts this foreign DNA, and integrates a __ at the leader end of the CRISPR locus 

diamonds

Repeats are represented as

rectangles 

spacers are represented as

protospacers

Regions of plasmids and viral DNA that were excised to become spacers are turned _

adjacent to short DNA sequence motifs

These protospacers are not randomly selected, if they were to become spacers they should be found ___ consisting of about 2 to 6 base pairs

protospacer adjacent motifs or PAM

• essential targeting component which distinguishes bacterial self from non-self DNA

• prevent the CRISPR locus from being targeted and destroyed by the CRISPR associated nucleus 

• component of the invading virus or plasmid BUT is not found in the bacterial host genome

• short DNA sequence motifs are termed __

pre crRNA that is processed into mature crRNAs

Subsequent viral infection or plasmid transfer will result in the transcription of CRISPR repeat spacer array into a _______ which are subsequently used as a guide by a Cas complex to interfere with the corresponding invading nucleic acid

not followed

Note that Cas9 will not successfully bind to or leave the target DNA sequence if it is __ by the PAM sequence

G G C G

PAM sequence

free crRNA

Subsequent infection with the same viral strain will result in the transcription of the CRISPR array yielding __ which will then be cleaved or cut to crRNAs

complex with cas enzymes

The specific crRNA will then form a ____ to cleave foreign DNA at sites containing the same sequence as the crRNA

functional guide RNA or a gRNA

In the CRISPR Cas9 system, the tracer RNA base pairs with the crRNA to form a __

directs the complex to a matching viral sequence

Cas9 uses the tracer RNA portion of the guide as a handle while the crRNA spacer sequence ___

Cas9 endonuclease

The ___ or variant thereof functions to bind to a guide RNA bind to target DNA in the presence of a guide RNA provided the target is upstream that is near the 5 prime end of a protospacer adjacent motif or PAM

Cas9 protein

__ is a dual RNA guided DNA endonuclease that uses the tracer RNA, crRNA duplex to direct DNA cleavage 

• responsible for introducing double strand breaks into invading plasmids and viruses

single guide RNA or sgRNA

The dual tracer RNA crRNA was engineered as a ___ that retains two critical features

• used to target or to edit any DNA sequence of interest as long as it is adjacent to a PAM 

2 critical features of sgRNA

1. The first feature retained is the 20 nucleotide sequence at the 5 prime end of the sgRNA that determines the DNA target site by Watson-Greek base pairing

2. The second feature is the double stranded structure at the the 3 prime side of the guide sequence that binds to Cas9

methods of delivering the CRISPR-Cas9 into the cell or embryo

1. expression plasmids via magnetofection

2. active protein via lipofection

3. mRNA encoding cas9 nucleus via lipofection

4. viral vectors encoding CRISPR components via magnetofection

Expression Plasmids

__ have sequences encoding Cas9 gene and single guide RNA and delivered via magnetofection

magnetofection

transfection of cells via magnetic nanoparticles complex with nucleic acids

Lipofection

__ is a lipid based transfection of cells, the protein is mixed with lipofection reagent and the complex is internalized by the cells via endocytosis

• Non-homologous end joining (NHEJ)

• Homology-directed repair (HDR)

When an engineered single guide RNA Cas9 introduces double strand DNA break at the target site, the cell employs 2 different DNA repair machinery namely __

Non-homologous end joining (NHEJ)

___ can result in random deletions or insertions at the repair site which may disrupt or alter gene functionality

Homology-directed repair

___ can result in gene correction or insertion by assisted recombination 

The prime editing system

__ introduces single stranded DNA breaks  instead of the double stranded DNA breaks

fusion protein

The prime editing system uses a __ consisting of a catalytically impaired Cas9 endonuclease fused to an engineered transcriptase enzyme and a prime editing guide RNA or pgRNA

pgRNA

__ capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides

prime editing tool advantages over CRISPR-Cas

• CRISPR-Cas9 edits rely on nonhomologous and joining or NHEJ or Homology-directed repair (HDR) to fix DNA breaks which could lead to unwanted random insertions or deletions

• prime editing system employs a different repair mechanism resulting in the retrieval of cells carrying the correct edit 

beta globin gene

CRISPR Cas was used by Chinese scientists to edit the __ in human embryos

inseminate mature oocytes

What these scientists did was to __ in a fertilization medium 4 hours after retrieval by conventional in vitro fertilization. Then, the fertilization status was checked 16 to 19 hours after insemination.

2 pronuclei

Normal fertilization was assessed by a presence of __ 1 pronucleus from the oocyte and the other from a sperm cell 

triploid

3 sets of chromosomes

microinjection

• Chinese scientists have also demonstrated the feasibility of gene editing in monkeys by introducing mutations in 2 different genes 

• In their experiment they delivered the Cas9 mRNA single guide RNA complex via __ into an embryo that was generated via insemination 

gene therapy

process of replacing defective genes with healthy ones or adding new genes to treat disease or help the body fight disease 

severe combined immunodeficiency (SCID)

• mutation in adenosine deaminase gene (ADA) 

Ashanti DeSilva

__ was the first person to be treated with gene therapy

Viruses

__ have been used as vectors to deliver therapeutic genes into human cells. These have been engineered to remove their ability to cause an infectious disease.

Luxturna

 a new gene therapy approved in 2017 to treat children and adult patients with an inherited form of vision loss that may result in blindness.

US FDA approved gene therapy products

• 2 of these products reprogram a patient’s own cells to attack a deadly cancer

• Luxturna

RPE65

Luxturna works by delivering a normal copy of the __ gene directly into the retinal cells. The retinal cells then produce the normal protein that converts light to an electrical signal in the retina to restore the patient's vision loss.

genetically engineered adeno-associated virus

Luxturna uses a __ as a vehicle to deliver the normal human RPE65 gene to the retinal cells to restore vision  

How is severe combined immunodeficiency or SCID treated using gene therapy ?

• First, a cloned normal copy of human adenosine deaminase gene (ADA) is excised from a plasmid and transferred into a viral vector which is then used to infect white blood cells removed from the patient.

• The transferred ADA gene is incorporated into a chromosome and becomes active.  

• After allowing the cells to grow and divide to increase their numbers, the cells are then reimplanted into the patient where they are expected to produce ADA which would allow the patient to develop an immune response