Jennifer A Thomson Department of Molecular and Cell Biology University of Cape Town South Africa

CRISPR Gene drive Bio-pharming Next generation DNA sequencing Quantitative real-time PCR Epigenetics

Clustered Regularly Interspersed Short Palindromic Repeats -----XX XX XX XX-- XX = PAM = sites where DNA can be cut by enzyme Cas9 (Protospacer Adjacent Motif) Cas9 = CRISPR-associated protein (number 9) Guide RNA = single stranded RNA version of DNA - guides Cas9 to correct XX Specific DNA site can be deleted/mutated  get rid of a trait eg. disease susceptibility OR New gene inserted  new trait eg. disease resistance

Cas9 = enzyme for cutting guide RNA PAM sequence = sites where DNA can be cut CRISPR  Mutations or Insertions

Clustered Regularly Interspersed Short Palindromic Repeats -----XX XX XX XX-- XX = PAM = sites where DNA can be cut by enzyme Cas9 (Protospacer Adjacent Motif) Cas9 = CRISPR-associated protein (number 9) Guide RNA = single stranded RNA version of DNA - guides Cas9 to correct XX Specific DNA site can be deleted/mutated  get rid of a trait eg. disease susceptibility OR New gene inserted  new trait eg. disease resistance

Cas9 CRISPR  Mutations or Insertions Target gRNA + Gene A

CRISPR  Mutations or Insertions Donor DNA  new gene A inserted Repair → mutations Gene A

CRISPR Gene drive Bio-pharming Next generation DNA sequencing Quantitative real-time PCR Epigenetics

Make transgenic mosquito which can’t carry malaria. BUT in wild gene diluted out by normal genes If include CRISPR/Cas9 with transgene the altered chromosome will “correct” wild chromosome  both carry transgene + CRISPR/Cas9 Release large numbers of altered mosquitoes  speed up process

Normal inheritance X Altered/new gene A Wildtype Gene A is diluted in subsequent generations A X X A

Make transgenic mosquito which can’t carry malaria. BUT in wild gene diluted out by normal genes If include CRISPR/Cas9 with transgene the altered chromosome will “correct” wild chromosome  both carry transgene + CRISPR/Cas9 Release large numbers of altered mosquitoes  speed up process

CRISPR/Gene drive inheritance X CRISPR/Cas9/new gene A Wildtype CRISPR/Cas9/A inherited A X X A A A A A

CRISPR Gene drive Bio-pharming Next generation DNA sequencing Quantitative real-time PCR Epigenetics

Conventional biologics manufacture: Stainless steel cell fermenters: large, expensive to set up, costly to run and to maintain

Credit: Stefan Schillberg, Fraunhofer IME medicines-in-plants-requires-new-regulations/ Biopharming uses the plant as factory

DNA coding for protein Introduce into plant by genetic engineering Formulate for Injection OR oral dosing Harvest plants, extract protein DOSE HUMANS OR ANIMALS OR transiently

A worker tends vegetables at the world's largest "plant factory" on July 2, The Japanese factory produces 10,000 heads of lettuce a day. Vertical farming: a perfect technology for pharmaceutical production

…two US healthcare workers who contracted Ebola in Liberia were treated with a cocktail of anti-Ebola Monoclonal Antibodies MADE IN PLANTS! Despite being given up to nine days post- infection in one case, it appears to have been effective Ed Rybicki, ViroBlogy 5 August as-therapy-for-ebola-in-humans-post-exposure-prophylaxis-goes-green/ Plant-made antibodies to the rescue – next time?

CRISPR Gene drive Bio-pharming Next generation DNA sequencing Quantitative real-time PCR Epigenetics

The generations of sequencing “1 st generation” Sanger di-deoxy capillary sequencing “2 nd (next) generation” 454/Roche, Illumina, ABI SOLiD “3 rd generation” Ion Torrent, PacBio, Oxford Nanopore ABI 3730/3730xl Illumina Genome Analyzer 454 FLX Titanium Ion Torrent PGM

Manual sequencing autoradiograms

Big Dye DNA sequencing Output from an automated DNA sequencing reaction – Each lane displays the sequence obtained from a separate DNA sample and primer

Computer reads the sequence

CRISPR Gene drive Bio-pharming Next generation DNA sequencing Quantitative real-time PCR Epigenetics

1. Detect the gene by PCR = Polymerase Chain Reaction DNA + enzyme DNA polymerase  amplification  visualise on gel 2. Detect the RNA by RT-PCR: Reverse Transcriptase PCR RNA + enzyme reverse transcription  DNA  PCR  gel 3. qRT-PCR: Quantitative Real Time (Reverse Transcriptase) PCR RT-PCR but with fluorescent tag incorporated  amount of RNA How to detect a specific gene; then how to detect its expression (RNA); then how to detect its RELATIVE expression (PCR  RT-PCR  qRT-PCR)

1. PCR AMPLIFICATION OF DNA

1. Detect the gene by PCR = Polymerase Chain Reaction DNA + enzyme DNA polymerase  amplification  visualise on gel 2. Detect the RNA by RT-PCR: Reverse Transcriptase PCR RNA + enzyme reverse transcription  DNA  PCR  gel 3. qRT-PCR: Quantitative Real Time (Reverse Transcriptase) PCR RT-PCR but with fluorescent tag incorporated  amount of RNA How to detect a specific gene; then how to detect its expression (RNA); then how to detect its RELATIVE expression (PCR  RT-PCR  qRT-PCR)

2. RT- PCR AMPLIFICATION OF RNA

1. Detect the gene by PCR = Polymerase Chain Reaction DNA + enzyme DNA polymerase  amplification  visualise on gel 2. Detect the RNA by RT-PCR: Reverse Transcriptase PCR RNA + enzyme reverse transcription  DNA  PCR  gel 3. qRT-PCR: Quantitative Real Time (Reverse Transcriptase) PCR RT-PCR but with fluorescent tag incorporated  amount of RNA How to detect a specific gene; then how to detect its expression (RNA); then how to detect its RELATIVE expression (PCR  RT-PCR  qRT-PCR)

Principle of Quantitative Amplification = measure time to reach threshold low copy target (e.g. ~10 3 copies) SIGNAL  (normalized fluorescence) TIME  ( amplification cycles) threshold of detection Real-Time Amplification Plot high copy target (e.g. ~10 8 copies) Initial lag phase no signal detected log phase signal ~doubles every cycle plateau phase reaction slows & stops as a component becomes limiting End point (not quantitative) Low = higher copy no. amplifies sooner High C T = lower copy no. amplifies later CTCT C T = threshold cycle (fractional cycle number where signal crosses a threshold of detection)

Relative Quantification

CRISPR Gene drive Bio-pharming Next generation DNA sequencing Quantitative real-time PCR Epigenetics

Epigenetics – ‘above/outside’ genetics Factors that change an organism’s characteristics without changing the DNA sequence DNA  RNA (message)  protein. Sum of proteins  organism’s characteristics Environment can  epigenetic changes  changes in organism’s characteristics Include smoking, eating, drinking, exercise (or lack), chemicals in air, water, food….

Epigenetics (cont.) What happens at DNA level: addition of methyl groups  turn genes on or off; packaging DNA regions into “bundles” which are inactive; opening DNA regions  active…. 20 YEARS AGO WE THOUGHT THESE CHANGES WERE NOT INHERITED. NOW WE KNOW THEY CAN BE! In humans – grandchildren may be more/less healthy due to our actions In plants – inheritance shown in next generation

CRISPR – particularly good for human/animal genetic engineering as can target changes  fewer “errors”. Plants can discard errors. But still is GMO though no “extra” DNA from vectors Gene drive – spread of changed organism in population Bio-pharming – cheaper, easier method of producing protein-based pharmaceuticals Next generation DNA sequencing – one of most important new technologies. BUT  data management and interpretation problems Quantitative real-time PCR – huge advantage for analysis of which genes are turned on/off (= expressed or not) when and under what conditions Epigenetics – how important for plants??? Nature vs nurture!