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

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

3. 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

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

5. 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

6. Cas9 CRISPR  Mutations or Insertions Target gRNA + Gene A

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

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

9. 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

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

11. 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

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

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

14. Conventional biologics manufacture: Stainless steel cell fermenters: large, expensive to set up, costly to run and to maintain http://www.theneweconomy.com/i40-2013

15. Credit: Stefan Schillberg, Fraunhofer IME https://www.jic.ac.uk/news/2013/02/growing- medicines-in-plants-requires-new-regulations/ Biopharming uses the plant as factory

16. 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

17. A worker tends vegetables at the world's largest "plant factory" on July 2, 2014. The Japanese factory produces 10,000 heads of lettuce a day. http://news.nationalgeographic.com/news/2014/07/140717-japan-largest-indoor-plant-factory-food/?sf3757507=1 Vertical farming: a perfect technology for pharmaceutical production

18. http://www.nbcnews.com/storyline/ebola-virus-outbreak/ebola-totally-out-control-doctors-without-borders-says-n136856

19. …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 2014 http://rybicki.wordpress.com/2014/08/05/plant-made-antibodies-used- as-therapy-for-ebola-in-humans-post-exposure-prophylaxis-goes-green/ Plant-made antibodies to the rescue – next time?

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

21. 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

22. Manual sequencing autoradiograms

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

24. Computer reads the sequence

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

26. 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)

27. 1. PCR AMPLIFICATION OF DNA

28. 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)

29. 2. RT- PCR AMPLIFICATION OF RNA

30. 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)

31. 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)

32. Relative Quantification

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

34. 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….

35. 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

36. 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!