Exam 3

GMOs, CRISPR, Base and Prime Editing, DNA fingerprinting, and the Flu

GMO stands for

genetically modified organism

GMO definition

organisms whose genetic material has been intentionally altered using biotechnology

GMOs can be generated by

• introducing mutations

• modifying endogenous genes

• silencing genes

• introducing foreign DNA

transgenic organism

a GMO that contains DNA originating from a different species

Modern GMOs include

• engineered human cells used in medicine and research

• crops

• pets

• microorganisms

• research animals

Public concerns of GM food

1. Human health

2. biodiversity

3. corporate control

4. environmental concerns

Large-scale cultivation of genetically uniform crops may

• reduce agricultural biodiversity

• favor monoculture systems

• increase vulnerability to pathogens

large biotechnology companies have historically dominated:

• seed patents

• herbicide systems

• GM crop commercialization

domination of large biotechnology companies leads critics to argue that it may

• concentrate economic power

• reduce farmer autonomy

• increase dependence on proprietary seed systems

ecological concerns of GM food

• spread of herbicide resistance

• Effects on non-target organisms

• altered ecosystem dynamics

• increased herbicide usage in some agricultural systems

round up ready crops

crops engineered to tolerate glyphosate

historically dominant traits in crops

• herbicide resistance

• insect resistance

glyphosate

active ingredient in Roundup herbicide that inhibits EPSPS

EPSPS

an enzyme required for the synthesis of aromatic amino acids in plants and many microorganisms

glyphosate remains the subject of

ongoing toxicological and regulatory debate worldwide

roundup ready crops express

a glyphosate-resistant bacterial EPSPS enzyme

the glyphosate-resistant bacterial EPSPS enzyme in Roundup Ready crops allows

crops to survive herbicide treatment

BT toxins express cry toxins derived from

the bacteria Bacillus thuringiensis

The proteins of the BT toxin damage

the digestive tract of susceptible insects, leading to insect death

Are BT toxins considered substantially more or less toxic to humans than many conventional insecticides

substantially less toxic to humans

BT toxins are highly specific towards

certain insect groups

foreign genes introduced into plants must be placed under the control of

compatible regulatory DNA sequences (including promoters and terminators)

many early GM crops used __ promotors

viral promotors

example of a viral promotor used in early GM crops

CaMV 35S promotor (became extremely widespread in plant biotechnology)

modern GM and gene-edited crops are increasingly engineered for

• drought tolerance

• salt tolerance

• disease resistance

• climate resilience

• longer shelf life

• improved nutritional value

traditional transgenic engineering introduces

foreign DNA into an organism

modern genome editing often tends to

modify only a few endogenous nucleotides

tools such as __ allow highly targeted modifications without necessarily introducing foreign genes

CRISPR-Cas9

the result of highly targeted modifications without necessarily introducing foreign genes

some modern engineered crops are genetically modified but not transgenic

processed foods commonly contain

DNA fragments and RNA molecules

Why is DNA rarely mentioned on food labels?

humans don’t use nucleic acids as a major energy source

GM ingredients are commonly detected using

PCR (polymerase chain reaction)

many GM crops contain shared engineered regulatory sequences such as

promotors, terminators, or transgene junctions

PCR primers targeting shared engineered regulatory sequences can

amplify GM-specific DNA fragments

quantitative PCR (qPCR)

estimates the relative abundance of GM material

largest producers of GM crops

U.S., Brazil, Argentina, Canada, and India

major GM crops

soybean, corn, cotton, and canola

strategies to genetically modify plants

• selective breeding

• polyploidy induction

• biolisitcs

• Agrobacterium tumefaciens

• RNA interference

• genome editing

how are larger polyploid plants produced

chemicals such as colchicine can disrupt microtubules and chromosome segregation

biolistics

“gene gun”, DNA-coated particles are physically shot into plant cells

Agrobacterium tumefaciens

bacterium that naturally transfers DNA into plant cells and is widely used as a gene delivery vector

plants can be introduced to produce __ that silence target genes

siRNAs

modern genome editing tools

• CRISPR

• Zinc-finger Nucleases

• TALENs

arctic apple

a genetically engineered apple designed to resist browning after cutting or bruising

polyphenol oxidase (PPO)

an enzyme involved in enzymatic browning reactions

The Arctic Apple produces RNA molecules that

suppress expression of polyphenol oxidase (PPO)

Why was the arctic apple notable?

it targeted consumer preference directly, rather than primarily benefiting agricultural production

older GMO regulations were largely written before

RNA interference, CRISPR, and modern genome editing technologies existed

modern genome editing can generate

subtle sequence changes, sometimes indistinguishable from natural mutations

Why do modern gene technologies that generate sequence changes indistinguishable from natural mutations create regulatory challenges?

because traditional GMO frameworks were designed mainly for clearly transgenic organisms containing foreign DNA

AquaAdvantage salmon

salmon that contains a growth-hormone transgene allowing faster growth and earlier market size, developed by AquaBounty, and received FDA approval after an unusually long regulatory process

Biotechnology is increasingly viewed as one potential tool to:

• Improve crop resilience

• Increase food production efficiency

• reduce agricultural losses

consequences of malnutrition and undernutrition

• impaired immune function

• developmental abnormalities

• reduced cognitive performance

• increased mortality

food insecurity remains disproportionally concentrated in

low income areas

__ convert food into biomass more efficiently than traditional livestock

insects

compared with cattle, insects generally require

• less water

• less land

• less feed

• produce fewer greenhouse gasses

De novo domestication

process of domesticating a wild plant species from scratch using modern genetic tools

innate potato

a genetically engineered potato that uses RNA interference (RNAi) to reduce the expression of certain genes

Environmental Protection Agency (EPA)

the U.S. Federal Agency responsible for protecting human health and the environment

Food and Drug Administration (FDA)

the U.S. federal agency responsible for ensuring the safety of food, drugs, cosmetics, and medical devices

United States Department of Agriculture (USDA)

U.S. federal department that regulates the import, movement, and field release of genetically engineered organisms

protoplast fusion

a technique in which the cell walls of two plant cells are removed enzymatically to produce protoplasts, which are then fused to create hybrid cells

polyploidy

the condition of having more than 2 complete sets of chromosomes in a cell or organism

genome editing

the targeted modification of the DNA sequence of a cell or organism

possible modifications that can be made to an organism/cell from genome editing

• inactivation of genes

• insertion of new DNA sequences

• correction of mutations

• precise editing of individual nucleotides

major genome editing technologies

• Zinc-finger nuclease (ZFN) - older

• TALENs - older

• meganucleases - older

• CRISPR-Cas system - newer

• base editors - newer

• prime editors - newer

ZFN, TALENs, and meganucleases predate CRISPR-based systems and were widely used before

the 2010s

most early genome editing systems function as

programmable molecular scissors

early genome editing systems contain a

DNA-recognition component physically linked to an endonuclease that cuts DNA

In ZFN, TALENs, and meganucleases, DNA recognition depends on

engineered proteins

In CRISPR-Cas systems, DNA recognition is guided by

RNA base pairing (makes retargeting dramatically easier)

base and prime editing are newer approaches designed to

• improve precision

• Reduce double-stranded breaks

• minimize unwanted mutations

What does CRISPR stand for

Clustered Regulatory Interspaced Short Palindromic Repeats

CRISPR systems defintion

natural defense mechanisms found in bacteria and archaea that protect prokaryotes from bacteriophages, plasmids, and other foreign genetic disorders

CRISPR functions as

an adaptive immune system

Steps of CRISPR systems in prokaryotes

1. Foreign DNA enters the cell, and fragments of the invading DNA can be integrated into the CRISPR locus

2. fragments are later transcribed into guide RNAs

3. guide RNAs associate with Cas proteins, which recognize and cleave matching foreign DNA during future infections

CRISPR-Cas9 requires

a guide RNA and the Cas9 endonucleases

Molecular steps of CRISPR-Cas9

1. guide RNA base pairs with complementary RNA target sequence

2. Cas9 generates a double stranded break in the DNA

3. cell repairs the DNA break (through one of two ways)

two ways the cell can repair the DNA break from the Cas9 protein

1. Non-homologous end joining (NHEJ)

2. Homology-directed repair (HDR)

Non-homologous end joining (NHEJ)

directly rejoins DNA ends from Cas9 protein break but is error prone

Non-homologous end joining (NHEJ) is useful for

gene knockout experiments

why is Non-homologous end joining (NHEJ) useful for gene knockout experiments?

it commonly introduces insertions, deletions, or frameshift mutations

Homology-directed repair (HDR)

method of repairing DNA break from the Cas9 protein that uses a homologous DNA template to repair the break

Homology-directed repair (HDR) compared to nonhomologous end joining (NHEJ)

HDR is more precise but less efficient than NHEJ

In homology-directed repair (HDR) researchers can

supply engineered donor DNA to insert sequences or correct mutation

base editor

modifies single DNA bases without generating double stranded DNA breaks

prime editing

more flexible system (than base editing) that is capable of introducing small insertions, deletions, and precise sequence changes

base and prime editing may

• Reduce off-target editing

• improve editing precision

hornless cattle

Naturally occurring hornless alleles can be introduced into dairy or beef cattle, reducing the need for dehorning procedures

hypoallergenic organisms

genome editing can remove genes encoding allergenic proteins (applications are hypoallergenic foods and improved xenotransplantation tissues)

genome editing can allow creation of

• more accurate disease models\

• miniature livestock models

• engineered primates

all of which improve biomedical research and antidrug testing

Disease-vector control could help

reduce diseases such as malaria, but ecological concerns remain substantial

researchers are attempting to introduce cold-adaptation traits from __ to __

from extinct mammoths to modern elephants (de-extinction)

primary medical goal of editing human genomes

treatment or prevention of genetic diseases

diseases that are current major targets of human genome editing

• sickle cell diseas

• B thalassemia

• cystic fibrosis

• duchenne muscular dystrophy

• some forms of blindless

• certain immune disorders

which diseases are currently the most tractable targets for human genome editing

diseases caused by single-gene mutations

human genome editing may target

• circulating blood cells

• somatic tissues

• stem cells

• embryos

• germline cells

somatic editing affects

only the treated individual (increasingly accepted clinically)

germline editing affects

sperm, eggs, or embryos, and can therefore be inherited by future generations (highly controversial and prohibited/heavily restricted in many countries

CRISPR machinery can include __ instead of a Cas protein

cas encoding DNA/RNA