![Page 1: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/1.jpg)
Cornerstones of CRISPR-Cas in Drug Discovery and Therapy
Swiss Pharma Science DayBern – August 22, 2017
Christof Fellmann, PhD Doudna Lab, UC Berkeley
![Page 2: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/2.jpg)
From Immune System to Precision Medicine
• Leveraging bacterial immunity for biomedicine• RNA‐guided precision genome engineering• CRISPR‐Cas for drug development• CRISPR‐Cas as ex‐vivo and in‐vivo therapeutic
![Page 3: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/3.jpg)
CRISPR‐Cas – A revolutionary genome engineering technology
The central dogma of molecular biology
• Unidirectional flow of genetic information• Medical approaches interact at the protein level• What if we could rewrite the genetic code?
![Page 4: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/4.jpg)
Leveraging bacterial immunity for biomedicine
Curiosity about how bacteria fight the flu
• RNA‐guided immune systems• RNAi, ZFN, TALEN• CRISPR‐Cas9
http://msms.ehe.osu.eduhttp://healtherandhappier.blogspot.comOrigamiwolf: https://flic.kr/p/oDCK1
Rodolphe Barrangou
![Page 5: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/5.jpg)
CRISPRs – Acquired immune systems in bacteria
Clustered Regularly Interspaced Short Palindromic Repeats
• Extrachromosomal and phage‐associated origins of spacers• Defense against bacteriophages• Streptococcus pyogenes (SpyCas9)
Ishino et al., 1987Bolotin et al., 2005Mojica et al., 2005Pourcel et al., 2005
Bacterial chromosome
CRISPR locus
![Page 6: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/6.jpg)
Bacterial CRISPR‐Cas adaptive immunity
Nuñez et al., Nature (2015a, 2015b), Nuñez et al., Mol Cell (2016)
How does RNA‐guidedDNA cleavage work?
![Page 7: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/7.jpg)
From Immune System to Precision Medicine
• Leveraging bacterial immunity for biomedicine• RNA‐guided precision genome engineering• CRISPR‐Cas for drug development• CRISPR‐Cas as ex‐vivo and in‐vivo therapeutic
![Page 8: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/8.jpg)
Programming Cas9 with single‐guide RNAs (sgRNAs)
CRISPR‐Cas9
• sgRNA guided• PAM for target specificity• Introduction of DNA DSBs
Jinek et al., Science 337, 816 (2012)
Cas9 programmed by sgRNA
![Page 9: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/9.jpg)
Cas9 and its guide RNA act like a molecular scalpel to cut DNA
Model: Jacob Corn; based on structure by Anders et al., Nature (2014).
RNA
DNA
Cas9 protein
![Page 10: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/10.jpg)
DNA DSB repair pathways – NHEJ, HDR (genome editing)
CRISPR‐Cas9, ZFN, TALEN
![Page 11: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/11.jpg)
From Immune System to Precision Medicine
• Leveraging bacterial immunity for biomedicine• RNA‐guided precision genome engineering• CRISPR‐Cas for drug development• CRISPR‐Cas as ex‐vivo and in‐vivo therapeutic
![Page 12: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/12.jpg)
CRISPR‐Cas genome engineering
Doudna and Charpentier, Science (2014)
Cas9 mRNA
sgRNA
+Cas9 RNP
Cas9
sgRNA
DNA
![Page 13: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/13.jpg)
CRISPR‐Cas as a tool for drug discovery – in cell culture
Fellmann et al., NRDD (2017)
• CRISPR‐Cas9• CRISPRi (interference)• CRISPRa (activation)
![Page 14: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/14.jpg)
Generation of a switch‐like (inducible) Cas9
Allosterically regulated Cas9
• Insertion of the estrogen receptor alpha ligand binding domain (ER‐LBD)• dCas9 transposition library• Assessment of large‐scale library in E. coli (CRISPRi screen)
Oakes, et al., Nat Biotech (2016)
![Page 15: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/15.jpg)
Generation of a switch‐like (inducible) Cas9
Allosterically regulated Cas9
• 4‐OH Tamoxifen regulated (like Cre‐ER system)• Validation in mammalian cells
Oakes, et al., Nat Biotech (2016)
![Page 16: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/16.jpg)
Inducible Cas9 – ArCas9
Allosterically regulated Cas9
• Serially inducible and reversible• GFP reporter cell line (BNL‐LMP‐15)
Oakes, et al., Nat Biotech (2016)
Cas9‐NLS
Cas9
ArCas9
![Page 17: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/17.jpg)
CRISPR‐Cas as a tool for drug discovery – animal models
Fellmann et al., NRDD (2017)
• CRISPR‐Cas9• CRISPRi (interference)• CRISPRa (activation)
![Page 18: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/18.jpg)
CRISPR‐Cas as a tool for drug discovery – animal models
Fellmann et al., NRDD (2017)
![Page 19: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/19.jpg)
From Immune System to Precision Medicine
• Leveraging bacterial immunity for biomedicine• RNA‐guided precision genome engineering• CRISPR‐Cas9 for drug development• CRISPR‐Cas9 as ex‐vivo and in‐vivo therapeutic
![Page 20: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/20.jpg)
Ex‐vivo genome editing – recoding primary human T cells
CXCR4, CCR5 editing forHIV treatment
Schumann, Lin et al., PNAS (2015).Shumann, Marson, Puck – UCSF
Martin – CHORICorn – Innovative Genomics Initiative
![Page 21: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/21.jpg)
A prudent path forward for genome engineering
What should we do now that genomes can be edited easily?
![Page 22: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/22.jpg)
A prudent path forward for genome engineering
![Page 23: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/23.jpg)
A prudent path forward for genome engineering
GENE EDITING
In vivo geneediting in dystrophicmousemuscleandmusclestem cellsMohammadshar i f Tabebordbar,1,2* Kexian Zhu,1,3* Jason K . W . Cheng,1
W ei Leong Chew,2,4 Jeffr ey J. W idr ick,5 W inston X . Yan,6,7 Clai r e M aesner ,1
El izabeth Y. W u,1† Ru X iao,8 F. Ann Ran,6,7 Le Cong,6,7 Feng Zhang,6,7
Luk H . Vandenberghe,8 George M . Church,4 Amy J. W agers1‡
GENE EDITING
Postnatal genomeeditingpartiallyrestoresdystrophin expression in amousemodel of muscular dystrophyChengzu Long,1,2,3* Leonela Amoasi i ,1,2,3* Alex A. M i reaul t,1,2,3 John R. M cAnal ly,1,2,3
Hui L i ,1,2,3 Efrain Sanchez-Or ti z,1,2,3 Samadr i ta Bhat tachar yya,1,2,3 John M . Shelton,4
Rhonda Bassel-Duby,1,2,3 Er ic N . Olson1,2,3†
GENE EDITING
In vivo genomeediting improvesmuscle function in amousemodel ofDuchennemuscular dystrophyChr istopher E. Nelson,1,2 Chady H . Hakim,3 David G. Ousterout,1,2
Prat iksha I . Thakore,1,2 Ei r ik A. M oreb,1,2 Ruth M . Castel lanos Rivera,4
Sar ina M adhavan,1,2 X iufang Pan,3 F. Ann Ran,5,6 W inston X . Yan,5,7,8
Aravind Asokan,4 Feng Zhang,5,9,10,11 Dongsheng Duan,3,12 Char les A. Gersbach1,2,13*
![Page 24: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/24.jpg)
A prudent path forward for genome engineering
What should we do now that genomes can be edited easily?
Baltimore, et al., Science (2015)
![Page 25: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/25.jpg)
From microbial immune system to precision medicine
Accelerate drug discovery and therapy
• CRISPR‐Cas editing, inhibition or activation (functional genomics)• Disease models• Cellular therapies
Fellmann et al., NRDD (2017)
![Page 26: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/26.jpg)
Acknowledgements
Doudna LabJennifer Doudna, PhDD‐Lab
![Page 27: Cornerstones of CRISPR-Cas in Drug Discovery and Therapy · Cornerstones of CRISPR-Cas in Drug Discovery and Therapy Swiss Pharma Science Day ... DNA DSB repair pathways –NHEJ,](https://reader036.vdocuments.us/reader036/viewer/2022070616/5d17c5b188c9938c2b8bb733/html5/thumbnails/27.jpg)
Thank you!