1. CRISPR Technology & Potential Implications
Saba Nafees

4. Overview Brief summary of mechanism History Current work
Implications & future work

5. CRISPR/CRISPR-associated (Cas) systems - overview
Acquired immunity evolved in bacteria and archaea
Clustered regularly interspaced short palindromic repeats
Use short RNA sequences to degrade foreign DNA
CRISPR locus - “effectively a genetic vaccination card in cells”- Jennifer Doudna
Similar to euk-RNA interference
Streptococcus pyogenes -> Cas9 protein

6. Overview cont’d Defense mechanism, one of many, against virus attacks
CRISPR/Cas System
Sequence is transcribed → small ‘guide’ RNA’s → guide Cas proteins to find and cleave future foreign threats
Host organism incorporates viral DNA into the CRISPR region of the genome

7. Overview cont’d - the locus

8. Overview cont’d - Cas gene & protein
Cas - CRISPR associated
Cas gene -> cas protein Nuclease → cutting DNA
Cas gene families -Cas I & Cas II found in all families of cas genes
3 mechanisms of CRISPR/Cas systems: ->acquire spacers and insert into host genome
Leader end - where foreign seqs are added

9. RNA-guided genetic silencing systems in bacteria and archaea - Weidenheft, Sternberg, Doudna, Nature, 2012

11. Overview cont’d

12. History -1987 : clustered repeats recognized by Japanese researchers
-iap gene in E. coli

13. History cont’d
-1990’s : clustered repeats with “strange sandwich characteristics” continued to be found in other species
-2002 : Ruud Jansen of Utrecht Univ. ϵ Netherlands called them CRISPR = cas genes located within 100’s of bp of crispr locus

14. History cont’d
-2005: 3 groups of scientists independently discovered adaptive immunity aspect (Koonin, Barrangou, Doudna)
-2007: UC Berkeley : Jennifer Doudna & colleagues
‘“Once we understood it as a programmable DNA- cutting enzyme, there was an interesting transition,” Doudna said. She and her colleagues realized there might be a very practical use for CRISPR. Doudna recalls thinking, “Oh my gosh, this could be a tool.”

15. Recent & Current -2013: Kevin Esvelt & George Church
-use of “custom” Guide RNA in human cells

16. Recent & Current cont’d
-2016 : David Liu and others
-dsDNA breaks -> random insertions or indels
-Base Editing: can be used for point mutations in human disease
r-crispr-advance-unveiled/

17. Commercialization
-many genome-editing companies have formed and continue to form
Ex:
Editas Medicine (G. Church, J. Doudna, D. Liu)
CRISPR Therapeutics (working on somatic cells, e.g., cftr gene in CF)
Desktop Genetics (gene-editing workflow software combining machine learning)

18. Diseases of the liver, eye, and lung. Delivery mechanisms established.
E.g.-hemoglobinopathies (sickle cell & beta thalassemia), some types of immunodeficiencies, immune approaches to cancer
Diseases of the liver, eye, and lung.
Delivery mechanisms established.

19. Implementation in mice embryonic stem cells
•CRISPR/Cas9-mediated simultaneous targeting of five genes in mES cells
•Generation of Tet1/Tet2 double-mutant mice in one step
•Generation of Tet1/Tet2 double-mutant mice with predefined mutations in one step
One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering, 2013

20. Genome editing into in-vitro fertilization
-indels & off- target mutations -> mosaic modification
-uncertain contributions of modified germline to the overall organism

21. In-vitro cont’d
Embryonic stem cell approach and zygote approach for genome editing-mediated gene correction to prevent a genetic disease. Zygotes with a mutation are treated with genome editing-mediated gene correction via embryonic stem cell approach or zygote approach. After embryo screening by preimplantation genetic diagnosis, one or more embryos which have a corrected gene with no off-target mutations are subjected to embryo transfer. NIPT can be used to confirm the genetic condition of the fetus. Subsequently, CVS or amniocentesis can confirm whether a fetus has genetic mosaic mutations. Long-term follow-up is required even after a successful birth owing to the contribution of the modified germline to the entire body. CVS: chorionic villus sampling, ESCs: embryonic stem cells, ET: embryo transfer, ICSI: intracytoplasmic sperm injection, IVF: in vitro fertilization, NIPT: non-invasive prenatal genetic testing, NT: nuclear transfer.

22. Ethics
A prudent path forward for genomic engineering and germline gene modification
-G.Church, J. Doudna, and others
At the dawn of the recombinant DNA era, the most important lesson learned was that public trust in science ultimately begins with and requires ongoing transparency and open discussion. That lesson is amplified today with the emergence of CRISPR-Cas9 technology and the imminent prospects for genome engineering. Initiating these fascinating and challenging discussions now will optimize the decisions society will make at the advent of a new era in biology and genetics.

23. Ethics cont’d Recommendations to:
“Strongly discourage germline modifications for clinical applications in humans” - must do so nationally & internationally
“Create forums in which experts can educate” - on scientific, legal, ethical, implications of genome modification
“Encourage & support transparent research”
“Convene globally representative group” -of experts, developers, etc., for further consideration & recommend policies

24. Questions
Origin of CRISPR/Cas system - was it casposons becoming friends?
What are other roles of this system? To use spacers as markers and recognizing cooperative bacteria?
Silencing self genes?