The CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 (CRISPR-associated protein 9) system is a targeted nuclease technology which allows precise genome editing. Since the discovery of this system there has been great interest in its potential for human gene therapy. The CRISPR-Cas9 system has many advantages in comparison to other targeted nucleases transcription activator-like effector nucleases and zinc finger nucleases. As a relatively new genome editing platform, safety issues such as off-target editing have yet to be fully investigated.This presentation addresses the challenges as well as the socio-ethical considerations that surround the use of human genome editing.

1. A New Molecular biology
technique for genome editing:
CRISPR-Cas9
HOW NEW DISCOVERY IS
CHANGING EVERYTHING AS
WE KNOW IT.
Vanessa Chappell
Cell Seminar 2016

2. Overview
• What is genome engineering and gene therapy?
• What molecular biology tools are available?
• What makes CRISPR a better option?
• Current research
• What does this mean for the future?

3. The basics:
.
Gene Therapy
Experimental techniques used to fix a
genetic problem at its source instead
of using a drug or surgery
Genome editing
DNA is inserted, deleted or replaced
in the genome of an organism using
engineered nucleases
Programmable Nucleases
DNA targeting platforms for genome
editing and gene therapy

4. Programmable Nucleases
Zinc-finger nucleases (ZFNs)
● 1st genomic editing strategy
● recognizes 3-4 bases of
sequence
● cost is high
● “finiky” to make
● need a new finger for each
study
Transcription Activator-Like
nucleases (TALENs)
● target a single nucleotide
● much larger than ZFN
● difficult to deliver
● fast to make
● cost is high (but less than ZFN)
Clusters Regularly
Interspaced Short Palidromic
Repeats (CRISPR)/CRISPR
associated (Cas9)
● uses a short guide RNA
● this complex is NOT manmade
● easily modified
● inexpensive
● efficient

5. Repair Mechanisms
(Thorne 2015)

6. What makes CRISPR/Cas so special?
What is it?
● bacterial adaptive
immunity
● can be easily modified
and programmed to
target any organism's
DNA
Where did it come from?
It was discovered in bacteria
when a researcher noticed
that the spaces in between
the CRISPRs matched the
DNA of virus’ that targeted
the bacteria
What does it do?
upon attack or initiation it
uses stored bits of RNA to
target and destroy an
invader

7. UAB stem cell
research lab 2016
The research team took stem cells from
a patient at Children’s of Alabama. They
converted these cells and used CRISPR-
Cas9 to target and correct the mutated
base pair. They were able to show that
the genes functioned normally using a
mouse model.
UAB researchers may be on their way to
curing sickle cell disease using CRISPR-
Cas9 technology.
***This research team is applying for
FDA permission to use what they have
found in the clinic on human patients.

9. (Wallpaper design by Art of the Cell)
Illustration of CRISPR-Cas9 Complex

10. Personalized Medicine
Babies born in Alabama hospitals currently are
tested for 35 diseases, “but for about the same price,
we could sequence their genome” and pinpoint
genes that put them at risk for disease, Townes says.
Then, “long before someone develops one of these
genetic predispositions, we could correct the gene
so that they never experience that disease,” Townes
says.

11. Summary
In general CRISPR/Cas9 is better than other engineered nucleases
because:
• generally more efficient at editing than ZFNs and TALENs
• CRISPR is RNA based so no need to reengineer the proteins to
recognize new DNA being studied (like the others)
• it can be multiplexed by co-transfection or co-injection of gRNA

12. FUTURE RESEARCH IS LIMITED ONLY BY YOUR
IMAGINATION

13. FUTURE RESEARCH
Medical
● treat genetic disease
● create specific antibiotics
● treat viral infections (HIV)
Agriculture
● GM plants
● pest resistant crops
● disease resistance livestock
Research
● study gene function
● target gene mutation
● create transgenic organisms
● synthetic biology

14. Why is it important?
Disease cure
● cystic fibrosis
● Huntington’s disease
● sickle-cell anemia
● HIV
● leukemia and other
blood disorders
● cancer
Agricultural
● better crops
● healthier livestock
● better breeding with
“gene drive”
Research
● faster methods
● less expensive
● more reliability

15. SETTING LIMITS ON WHAT SHOULD BE
ALLOWED
• Barcode babies
• Smart genes
• Skinny genes
• Pretty genes
• What’s next??

16. References
Ahmandi, Maryam. 2016. "Utilization of Site-Specific Recombination in Biopharmaceutical Production." Iranian Biomedical Journal 68-76.
Auffray, charles, Timothy Caulifield, Julian L Griffin, Mulin Khoury, James R Lupski, and et al. 2016. "From genomic medicine to precision medicine; highlights of 2015."
Genome Medicine 8.
Boon, Reinier A, Nicholas Jae, Lesca Holdt, and Stephanie Dimmeler. 2016. "Long Noncoding RNAs; From Clinical Genetics to Therapeutic Targets?" Journal of the American
College of cardiology 1214-1226.
Cong, Le. 2013. "Multiplex Genome Engineering Using CRISPR/Cas System." Science 819-823.
Editor, Staff News. 2016. "Science; Study Results from Wellcome Trust Sanger Institute Update Understanding of Science [CRISPR-Cas9 (D10A) nickase-based genotypic and
phenotypic screening to enhance genome editing]." Health and Medicine Week 3283.
Findlay, S.D., Vincent, K.M., Berman, J.R., & Postovit, L. 2016. "A digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells." Plos One
(Public Library of Science) 11(4).
Fodil, Nassima, David Langlais, and Philippe Gros. 2016. "Primary Immunodeficiencies and Inflammatory Disease: A Growing Genetic Intersection." Trends in Immunology
126-140.
Gaj, Thomas. 2013. "ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering." Cell: Trends in Biotechnology 397-405.
Jakociunas, Tadas. 2015. "Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae." Elsevier 213-222.
Jo, Young-Il. 2015. "CRISPR/Cas9 system as an innovative genetic engineering tool: Enhancements in sequence specificity and delivery methods." Elsevier 234-243.
Kaczmarczyk, Lech, Ylva Mende, Branko Zevnik, and Walker Jackson. 2016. "Manipulating the Prion Protein Gene Sequence and Expression Levels with CRISPR/Cas9." Plos
One 11(4).
Kennedy, Edward M. 2015. "Optimization of a mulitiplex CRISPR/Cas system for use as an antiviral theraputic." Elsevier 82-86.
Kuritzkes, Daniel R. 2016. "Hematopoietic stem cell transplantation for HIV cure." Journal of Clinical Investigation 432-437.
Mougiakos, Ioannis. 2016. "Next Generation Prokaryotic Engineering: The CRISPR-Cas Toolkit." Trends in Biology 13.
Shiraz A. Shah, Gisle Vestergaard, and Roger A. Garrett. 2012. "Chapter 9: CRISPR/Cas and CRISPR/Cmr Immune Systems of Archaea." In Regulatory RNAs in Prokaryotes, by
Wolfgang R. Hess and Anita Marchfelder, 163-181. Springer Vienna.

17. “
”
“The first promise of any good politician is to make people's lives
better and scientific research leading to Innovation is one of the
best ways to honor that promise. Until about 1700, there was
basically no development. Almost everybody was poor. Many
were sick. One of every 4 children died. The average lifespan
was about 40 years and 99% of people were illiterate. But then
science came along and we started inventing---electricity,
steam engine, antibiotics, sanitation, vaccines, microprocessors
and genetic medicine…”
Bill Gates, during a speech about how political leadership can accelerate innovation.
Science is the Great Giver--and we're just at the
beginning of what it can give. “

18. QUESTIONS??