Posts Tagged ‘Sickle Cell disease’

Ribozymes and RNA Machines –  Work of Jennifer A. Doudna

Reporter: Aviva Lev-Ari, PhD, RN


UPDATED 3/27/2014

New DNA-editing technology spawns bold UC initiative

Crispr Goes Global

UPDATED 3/5/2014

Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity

One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering

RNA-Guided Human Genome Engineering via Cas9


From: Expert CRISPR/Cas9 Publications <>
Date: Tue, 04 Mar 2014 17:03:01 +0000
To: <>
Subject: CRISPR-mediated gene editing resources


UPDATED on 11/10/2013

Exclusive: ‘Jaw-dropping’ breakthrough hailed as landmark in fight against hereditary diseases as Crispr technique heralds genetic revolution

Development to revolutionise study and treatment of a range of diseases from cancer, incurable viruses such as HIV to inherited genetic disorders such as sickle-cell anaemia and Huntington’s disease


Thursday 07 November 2013

A breakthrough in genetics – described as “jaw-dropping” by one Nobel scientist – has created intense excitement among DNA experts around the world who believe the discovery will transform their ability to edit the genomes of all living organisms, including humans.

Click image above to enlarge graphic

The development has been hailed as a milestone in medical science because it promises to revolutionise the study and treatment of a range of diseases, from cancer and incurable viruses to inherited genetic disorders such as sickle-cell anaemia and Down syndrome.

For the first time, scientists are able to engineer any part of the human genome with extreme precision using a revolutionary new technique called Crispr, which has been likened to editing the individual letters on any chosen page of an encyclopedia without creating spelling mistakes. The landmark development means it is now possible to make the most accurate and detailed alterations to any specific position on the DNA of the 23 pairs of human chromosomes without introducing unintended mutations or flaws, scientists said.

The technique is so accurate that scientists believe it will soon be used in gene-therapy trials on humans to treat incurable viruses such as HIV or currently untreatable genetic disorders such as Huntington’s disease. It might also be used controversially to correct gene defects in human IVF embryos, scientists said.

Until now, gene therapy has had largely to rely on highly inaccurate methods of editing the genome, often involving modified viruses that insert DNA at random into the genome – considered too risky for many patients.

The new method, however, transforms genetic engineering because it is simple and easy to edit any desired part of the DNA molecule, right down to the individual chemical building-blocks or nucleotides that make up the genetic alphabet, researchers said.

“Crispr is absolutely huge. It’s incredibly powerful and it has many applications, from agriculture to potential gene therapy in humans,” said Craig Mello of the University of Massachusetts Medical School, who shared the 2006 Nobel Prize for medicine for a previous genetic discovery called RNA interference.

“This is really a triumph of basic science and in many ways it’s better than RNA interference. It’s a tremendous breakthrough with huge implications for molecular genetics. It’s a real game-changer,” Professor Mello told The Independent.

“It’s one of those things that you have to see to believe. I read the scientific papers like everyone else but when I saw it working in my own lab, my jaw dropped. A total novice in my lab got it to work,” Professor Mello said.

In addition to engineering the genes of plants and animals, which could accelerate the development of GM crops and livestock, the Crispr technique dramatically “lowers the threshold” for carrying out “germline” gene therapy on human IVF embryos, Professor Mello added.

The new method of gene therapy makes it simple and easy to edit any desired part of the DNA molecule (Getty Creative)

The new method of gene therapy makes it simple and easy to edit any desired part of the DNA molecule (Getty Creative) Germline gene therapy on sperm, eggs or embryos to eliminate inherited diseases alters the DNA of all subsequent generations, but fears over its safety, and the prospect of so-called “designer babies”, has led to it being made illegal in Britain and many other countries.

The new gene-editing technique could address many of the safety concerns because it is so accurate. Some scientists now believe it is only a matter of time before IVF doctors suggest that it could be used to eliminate genetic diseases from affected families by changing an embryo’s DNA before implanting it into the womb.

“If this new technique succeeds in allowing perfectly targeted correction of abnormal genes, eliminating safety concerns, then the exciting prospect is that treatments could be developed and applied to the germline, ridding families and all their descendants of devastating inherited disorders,” said Dagan Wells, an IVF scientist at Oxford University.

“It would be difficult to argue against using it if it can be shown to be as safe, reliable and effective as it appears to be. Who would condemn a child to terrible suffering and perhaps an early death when a therapy exists, capable of repairing the problem?” Dr Wells said.

The Crispr process was first identified as a natural immune defence used by bacteria against invading viruses. Last year, however, scientists led by Jennifer Doudna at the University of California, Berkeley, published a seminal study showing that Crispr can be used to target any region of a genome with extreme precision with the aid of a DNA-cutting enzyme called CAS9.

Since then, several teams of scientists showed that the Crispr-CAS9 system used by Professor Doudna could be adapted to work on a range of life forms, from plants and nematode worms to fruit flies and laboratory mice.

Earlier this year, several teams of scientists demonstrated that it can also be used accurately to engineer the DNA of mouse embryos and even human stem cells grown in culture. Geneticists were astounded by how easy, accurate and effective it is at altering the genetic code of any life form – and they immediately realised the therapeutic potential for medicine.

“The efficiency and ease of use is completely unprecedented. I’m jumping out of my skin with excitement,” said George Church, a geneticist at Harvard University who led one of the teams that used Crispr to edit the human genome for the first time.

“The new technology should permit alterations of serious genetic disorders. This could be done, in principle, at any stage of development from sperm and egg cells and IVF embryos up to the irreversible stages of the disease,” Professor Church said.

David Adams, a DNA scientist at the Wellcome Trust Sanger Institute in Cambridge, said that the technique has the potential to transform the way scientists are able to manipulate the genes of all living organisms, especially patients with inherited diseases, cancer or lifelong HIV infection.

“This is the first time we’ve been able to edit the genome efficiently and precisely and at a scale that means individual patient mutations can be corrected,” Dr Adams said.

“There have been other technologies for editing the genome but they all leave a ‘scar’ behind or foreign DNA in the genome. This leaves no scars behind and you can change the individual nucleotides of DNA – the ‘letters’ of the genetic textbook – without any other unwanted changes,” he said.

Timeline: Landmarks in DNA science

Restriction enzymes: allowed scientists to cut the DNA molecule at or near a recognised genetic sequence. The enzymes work well in microbes but are more difficult to target in the more complex genomes of plants and animals. Their discovery in the 1970s opened the way for the age of genetic engineering, from GM crops to GM animals, and led to the 1978 Nobel Prize for medicine.

Polymerase chain reaction (PCR): a technique developed in 1983 by Kary Mullis (below, credit: Getty) in California allowed scientists to amplify the smallest amounts of DNA – down to a single molecule – to virtually unlimited quantities. It quickly became a standard technique, especially in forensic science, where it is used routinely in analysing the smallest tissue samples left at crime scenes. Many historical crimes have since been solved with the help of the PCR test. Mullis won the Nobel Prize for chemistry in 1993.

RNA interference: scientists working on the changing colour of petunia plants first noticed this phenomenon, which was later shown to involve RNA, a molecular cousin to DNA. In 1998, Craig Mello and Andrew Fire in the US demonstrated the phenomenon on nematode worms, showing that small strands of RNA could be used to turn down the activity of genes, rather like a dimmer switch. They shared the 2006 Nobel Prize for physiology or medicine for the discovery.

Zinc fingers: complex proteins called zinc fingers, first used on mice in 1994, can cut DNA at selected sites in the genome, with the help of enzymes. Another DNA-cutting technique called Talens can do something similar. But both are cumbersome to use and difficult to operate in practice – unlike the Crispr technique.


a video of how the Crispr system derived from bacteria works on human cells to correct genetic defects


Jennifer A. Doudna

Professor of Chemistry
Professor of Biochemistry & Molecular Biology

office: 708A Stanley Hall
phone: 510-643-0225
fax: 510-643-0008

lab: 731 Stanley Hall
lab phone: 510-643-0113
lab fax: 510-643-0080

Research Group URL
Recent Publications

Research Interests

Chemical Biology

Ribozymes and RNA Machines: RNA forms a variety of complex globular structures, some of which function like enzymes or form functional complexes with proteins. There are three major areas of focus in the lab: catalytic RNA, the function of RNA in the signal recognition particle and the mechanism of RNA-mediated internal initiation of protein synthesis. We are interested in understanding and comparing catalytic strategies used by RNA to those of protein enzymes, focusing on self-splicing introns and the self-cleaving RNA from hepatitis delta virus (HDV), a human pathogen. We are also investigating RNA-mediated initiation of protein synthesis, focusing on the internal ribosome entry site (IRES) RNA from Hepatitis C virus. Cryo-EM, x-ray crystallography and biochemical experiments are focused on understanding the structure and mechanism of the IRES and its amazing ability to hijack the mammalian ribosome and associated translation factors. A third area of focus in the lab is the signal recognition particle, which contains a highly conserved RNA required for targeting proteins for export out of cells. Each of these projects seeks to understand the molecular basis for RNA function, using a combination of structural, biophysical and biochemical approaches.


Medical School, 1989-1991; Post-doctoral fellow, University of Colorado, 1991-1994; Assistant/Associate professor, (1994-1998), Professor, (1999-2001), Yale University. Professor of Biochemistry & Molecular Biology, UC Berkeley, (2002-). Howard Hughes Medical Investigator 1997 to present. Packard Foundation Fellow Award, 1996; NSF Alan T. Waterman Award, 2000. Member, National Academy of Sciences, 2002. Member, American Academy of Arts and Sciences, 2003; American Association for the Advancement of Science Fellow Award, 2008; Member, Institute of Medicine of the National Academies, 2010.


Diagnosing Diseases & Gene Therapy: Precision Genome Editing and Cost-effective microRNA Profiling


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Author and Reporter: Anamika Sarkar, Ph.D.

Nitric Oxide (NO) is highly regulated in the blood such that it can be released as vasodilator when needed. The importance and pathway of Nitric Oxide has been nicely reviewed by. “Discovery of NO and its effects of vascular biology”. Other articles which are good readings for the importance of NO are  – a) regulation of glycolysis b) NO in cardiovascular disease c) NO and Immune responses Part I and Part II d) NO signaling pathways. The  effects of NO in diseased states have been reviewed by the articles – “Crucial role of Nitric Oxide in Cancer”, “Nitric Oxide and Sepsis, Hemodynamic Collapse, and the Search for Therapeutic Options”.. (Also, please see Source for more articles on NO and its significance).

Computational models are very efficient tools to understand complex reactions like NO towards physiological conditions. Among them wall shear stress is one of the major factors which is reviewed in the article – “Differential Distribution of Nitric Oxide – A 3-D Mathematical Model”.

Moreover, decrease in availability of NO can lead to many complications like pulmonary hypertension. Some of the causes of decrease in NO have been identified as clinical hypertension, right ventricular overload which can lead to cardiac heart failure, low levels of zinc and high levels of cardiac necrosis.

Sickle Cell disease patients, a hereditary disease, are also known to have decreased levels of NO which can become physiologically challenging. In USA alone, there are 90,000 people who are affected by Sickle cell disease.

Sickle cell disease is breakage of red blood cells (RBC) membrane and resulting release of the hemoglobin (Hb) into blood plasma. This process is also known as Hemolysis. Sickle cell disease is caused by single mutation of Hb which changes RBC from round shape to sickle or crescent shapes (Figure 1).


Figure 1 (A) shows normal red blood cells flowing freely through veins. The inset shows a cross section of a normal red blood cell with normal hemoglobin. Figure 1 (B) shows abnormal, sickled red blood cells The inset image shows a cross-section of a sickle cell with long polymerized HbS strands stretching and distorting the cell shape. Image Source:

Sickle Cell RBCs has much shorter life span of 10-20 days when compared with normal RBCs 100-120 days lifespan. Shorter life span of Sickle cell disease RBC’s are compensated by bone marrow generation of new RBCs. However, many times new blood generation cannot cope with the small life span of Sickle cell RBCs and causes pathological condition of Anemia.

RBCs generally breakdown and release Hbs in blood plasma after they reach their end of life span. Thus, in case of Sickle cell disease, there is more cell free Hb than normal. Furthermore, it is known that NO has a very high affinity towards Hbs, which is one of the ways free NO is regulated in blood. As a result presence of larger amounts of cell free Hb in Sickle cell disease lead to less availability of NO.

However, the question remained “what is the quantitative relationship between cell free Hb and depletion of NO. Deonikar and Kavdia (J. Appl. Physiol., 2012) addressed this question by developing a 2 dimensional Mathematical Model of a single idealized arteriole, with different layers of blood vessels diffusing nutrients to tissue layers (Figure 2:  Deonikar and Kavdia Figure 1).


cell free Hb in 2 dimensional representations of blood vessels.

The authors used steady state partial differential equation of circular geometry to represent diffusion of NO in blood and in tissues. They used first and second order biochemical reactions to represent the reactions between NO and RBC and NO autooxidation processes. Some of their reaction model parameters were obtained from literature, rest of them were fitted to experimental results from literature. The model and its parameters are explained in the previously published paper by same authors Deonikar and Kavdia, Annals of Biomed., 2010. The authors found that the reaction rate between NO and RBC is 0.2 x 105, M-1 s-1 than 1.4 x 105, M-1 s-1 as reported before by Butler, Biochim. Biophys. Acta, 1998.

Their results show that even small increase in cell free Hb, 0.5uM, can decrease NO concentrations by 3-7 folds approximately (comparing Fig1(b) and 1(d) of Deonikar and Kavdia, 2012, as shown in Figure 2 of this article). Moreover, their mathematical analysis shows that the increase in diffusion resistance of NO from vascular lumen to cell free zone has no effect on NO distribution and concentration with available levels of cell free Hb.

Deonikar and Kavdia’s mathematical model is a simple representation of actual physiological scenario. However, their model results show that for Sickle cell disease patients, decrease in levels of bioavailable NO is an attribute to cell free Hb, which is in abundant for these patients. Their results show that small increase by 0.5 uM in cell free Hb can cause large decrease in NO concentrations.

These interesting insights from the model can help in further understanding in the context of physiological conditions, by replicating experiments in-vivo and then relating them to other known diseases of Sickle cell disease patients like Anemia, Pulmonary Hypertension. Further, drugs can be targeted towards decreasing free cell Hbs to keep balance in availability of NO, which in turn may help in other related disease like Pulmonary Hypertension of Sickle Cell disease patients.


Deonikar and Kavdia (2012) :

Previous model explaining mathematical representation and parameters used in the model :Deonikar and Kavdia, Annals of Biomed., 2010.

Previous paper stating reaction rate of Hb and NO: Butler, Biochim. Biophys. Acta, 1998.

Causes of decrease in NO

Clinical Hypertension :

Right ventricular overload :

Low levels of zinc and high levels of cardiac necrosis :

Sickle Cell Source:

NO Source:

Differential Distribution of Nitric Oxide – A 3-D Mathematical Model:

Discovery of NO and its effects of vascular biology

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

Nitric oxide: role in Cardiovascular health and disease

NO signaling pathways

Nitric Oxide and Immune Responses: Part 1

Nitric Oxide and Immune Responses: Part 2

Statins’ Nonlipid Effects on Vascular Endothelium through eNOS Activation

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography

Nitric Oxide, Platelets, Endothelium and Hemostasis

Crucial role of Nitric Oxide in Cancer

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure

Nitric Oxide and Sepsis, Hemodynamic Collapse, and the Search for Therapeutic Options

NO Nutritional remedies for hypertension and atherosclerosis. It’s 12 am: do you know where your electrons are?

Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination?

Endothelial Function and Cardiovascular Disease

Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Endothelial Dysfunction, Diminished Availability of cEPCs,  Increasing  CVD Risk – Macrovascular Disease – Therapeutic Potential of cEPCs

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production


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