A New Molecular biology technique for genome editing: CRISPR-Cas9

HOW NEW DISCOVERY IS

CHANGING EVERYTHING AS

WE KNOW IT.

Vanessa ChappellCell Seminar 2016

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?

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

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

Repair Mechanisms

(Thorne 2015)

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

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.

(Wallpaper design by Art of the Cell)

Illustration of CRISPR-Cas9 Complex

Personalized MedicineBabies 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.

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

FUTURE RESEARCH IS LIMITED ONLY BY YOUR IMAGINATION

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

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

SETTING LIMITS ON WHAT SHOULD BE ALLOWED

• Barcode babies• Smart genes• Skinny genes• Pretty genes• What’s next??

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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

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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.

“

”“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. “

QUESTIONS??