Ag Biotech + COVID 1

why is ag biotech necessary

925 million people undernourished (FAO 2010), including 171 million children globally stunted by malnutrition. As many as 15 million may die of starvation per year compared to 7.6 million who die of cancer. Even newer lower population estimates predict 8.4 billion population by 2030 (an increase of 25%). Many millions expecting increase food quantity/quality. We will need more fresh water than may be available.

corn borer

Worms eat the ears (where the corn is) and can destroy the stem. Leads to secondary fungal infections as well.

Bt corn

Bacillus thuringiensi (Bt) is a species of bacteria that synthesizes a protein (Cry) toxic to certain classes of insects (corn borers). Cry protein sprayed on corn kills the corn borer insect and increases yield. But… expensive to make, store and transport; uneven application; broad spraying can kill beneficial insects

What if the corn plants made their own Cry protein? What would you need to do to turn the Bt Cry gene into one that would work in corn?

Add corn promoter, enhancers, transcription terminator. Add 5’UTR sequences with the start AUG as the first AUG. Add 3’UTR sequences that allow for transcript stability. Can have the same coding sequences (bacteria and corn have the same genetic code)

Use CRISPR to transform corn and insert modified Cry2 gene. Corn will now express Cry2 in locations based on the enhancers you chose, and the protein will go where you direct it.

what is golden rice made to do

Golden rice is engineered to synthesize beta-carotene (Vitamin A precursor). Rice normally makes beta-carotene, but only in photosynthetic tissues, not in developing seeds. So, the genes encoding the biosynthetic enzymes are present in the rice genome, but not expressed in developing seed cells

Need a seed enhancer bound by a Reg TF to get the beta-carotene gene expressed in seed cells

what do seed enhancers do

seed enhancers are only active in seed cells. In an existing rice gene, you swap out the enhancer for a seed enhancer to get that gene active in seeds

find seed enhancers by putting them next to reporter genes and see if that gene gets activated in the seeds

different engineered traits in GMO crops

Engineered traits:

– herbicide resistance

– insect resistance

– drought tolerance

– viral resistance

– increased nutritional value

what are viruses

Viruses are parasites that are everywhere

Viruses are not organisms -- they are particles or agents; they are obligate parasites. Viruses have a genome (RNA or DNA) and they evolve. Viruses enter a host cell and take over its biosynthetic machinery – viruses are not alive because are absolutely dependent on the host cell machinery.

Virus genomes and evolution

Viral genomes are DNA or RNA / double- or single-stranded

Evolution acts on DNA variation to select for organisms that have higher fitness (more successful) than their competitors, leading to changes in genomes over time. For viruses, selection goes on at turbo speeds:

1. Viruses compete is vast numbers, so even a tiny, tiny advantage can win out

2. Viruses typically make many more mistakes than other organisms during the process of replication – i.e., much more genome variation

3. Viruses have very rapid replication time (hours) – many generations per unit time

4. Viruses are essential a genome in a shell (protein or membrane). Thus, the size of the genome has a huge impact on fitness (smaller = more fit)

human genome vs virus genome

Most of the genome in many eukaryotic organisms is parasitic junk. Only a small part is coding. Viral genomes are incredibly compact. There are no wasted sequences in viral genomes!!

Many viruses have evolved overlapping reading frames – two proteins are encoded by a segment of DNA read in different reading frames. Overlapping reading frames use a single sequence for two (or even three) protein coding reading frames

how viral genomes maximize protein production besides overlapping reading frames

Proteolytic cleavage: Multiple proteins encoded by a single open reading frame: the long polypeptide must be cut by a protease to release the different proteins. One open reading frame translated into one protein, then individual proteins released by proteases

What’s the relationship between overlapping reading frames and proteolytic cleavage

Both are evolutionary adaptations that lead to a smaller viral genome size providing a competitive advantage (improved fitness). The mechanisms that lead to overlapping reading frames and proteolytic cleavage are completely different. A specific virus might deploy neither, both or one of these strategies

SARS-2: Both (primarily proteolytic cleavage, but also a bit of overlapping reading frames)

lytic cycle

Lytic lifecycle: Viral genome enters host

cell. Viral genes get transcribed and translated. Viral genome replicated. Viruses released, killing the host cell. Infection spreads to other cells or other hosts

Lysogenic cycle

Lysogenic lifecycle: Viral genome enters host cell. Viral genome inserted* into host genome. When host cell divides, viral genome is replicated as well. In non-dividing cells, some lysogenic viruses simply package their genomes into heterochromatin without genome insertion

A period of quiescence/latency

Viral sequences in each cell can later be reactivated to produce viral particles (this is part of the lysogenic cycle)

ex: Once the HIV genome is inserted into the T cell genome, it can remain quiescent for decades

herpes lifecycle

Herpes virus infects epithelial cells (at an injury site)

Lytic cycle in epithelial cell. Released viruses infect sensory neurons. Lysogenic cycle in the neuronal cell. Reactivation occurs when the neuron makes a few viral particles (neuron survives). Lytic infection in epithelial cells (herpes outbreak)

Recognizing virally-infected cells

When a virus is engaged in virus replication (either in a lytic cycle or the activated portion of the lysogenic cycle), viral particles are presented on the surface of the cells. These are recognized by your immune system. The infected cell is killed, preventing viral replication

Why is it that lysogenic viral infections are nearly impossible to “cure”?

The immune system cannot recognize the cell; There are no viral proteins to target with drugs in latent cells

coronavirus

Coronaviruses are a large family of viruses that infect animals and humans. Current “novel coronavirus” is named SARS-CoV-2 or SARS-2. COVID-19 refers to the human disease state

+ssRNA genome, Membrane envelope (from human host cell), spike glycoprotein allows entry into human cells

COVID host cell entry

The human protein ACE2 helps regulate blood pressure normally and is found on the cell surface of a subset of airway epithelial cells. The SARS-2 Spike protein binds to ACE2. Once bound, the virus is endocytosed into the host cell (engulfed and brought into the cell)

spike protein

RBD: Binds to the human ACE2 receptor

Furin cleavage site: sequence recognized by a human protease

long end of spike provide the “engine” for membrane fusion and viral entry, and transmembrane domain at end

SARS-2 infection

The SARS-2 Spike protein binds to the human ACE2 protein on cells that express it. SARS-2 can only infect ACE2-expressing cells. Binding triggers viral endocytosis. The Furin cleavage site is cleaved by a human protease, releasing the “head group” and allowing the fusion machinery to function efficiently. Without the Furin cleavage site, SARS-2 is not infectious in humans. Once the virus fuses its membrane with the endocytic membrane, the viral genome is released into the cytoplasm

Viral entry and translation

The SAR-2 genome is a “+” strand RNA genome (single-stranded). The “+” genome refers to the RNA strand that contains the open reading frame for proteins (like an mRNA). The viral +RNA genome is immediately translated upon entry into the cytoplasm. Viral proteins are produced

viral genome replication

Because the viral genome is ssRNA, it needs to be copied into a complementary strand for further genomic copies to be made. The viral positive (+) strand ssRNA genome is copied into a complementary negative (-) RNA strand by the viral RNA-dependent-RNA-polymerase protein (RdRp).

what are + and - strands used for

The negative strand RNA is then used as a template to synthesize many more viral + strand RNAs, and the + strand mRNAs are used both for translation of viral proteins and as the genomes of new viral particles using “stolen” endomembrane envelopes to package