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From: Expert CRISPR/Cas9 Publications <Expert_CRISPRCas9_Publications@mail.vresp.com>
Date: Tue, 04 Mar 2014 17:03:01 +0000
To: <avivalev-ari@alum.berkeley.edu>
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

STEVE CONNOR

SCIENCE EDITOR

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) 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.

VIEW VIDEO

http://www.independent.co.uk/news/science/indyplus-video-crispr-technique-8925604.html

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

SOURCE

http://www.independent.co.uk/news/science/exclusive-jawdropping-breakthrough-hailed-as-landmark-in-fight-against-hereditary-diseases-as-crispr-technique-heralds-genetic-revolution-8925295.html?goback=%2Egde_2106240_member_5804987154979381248#%21

Jennifer A. Doudna

Professor of Chemistry
Professor of Biochemistry & Molecular Biology

email: doudna@berkeley.edu
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.

Biography

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.

READ MORE

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

http://pharmaceuticalintelligence.com/2013/03/28/diagnosing-diseases-gene-therapy-precision-genome-editing-and-cost-effective-microrna-profiling/

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Sexed Semen and Embryo Selection in Human Reproduction and Fertility Treatment

Posted in Biological Networks, Gene Regulation and Evolution, Genome Biology, Genomic Testing: Methodology for Diagnosis, Medical and Population Genetics, Personalized and Precision Medicine & Genomic Research, Pharmacogenomics, Population Health Management, Genetics & Pharmaceutical, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Reproductive Biology & Bio Instrumentation, tagged chromosome, DNA, embryo selection, human reproduction, ICSI, In vitro fertilisation, Intracytoplasmic sperm injection, IVF, PGS, Pregnancy rate, sexed semen, Sperm sorting, XY on March 11, 2013| 4 Comments »

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

Use of sexed semen in conjunction with in vitro embryo production is a potentially efficient means of obtaining offspring of predetermined sex. Sperm sorting is a means of choosing what type of sperm cell is to fertilize the egg cell. It can be used to sort out sperm that are most healthy, as well as determination of more specific traits, such as sex selection in which spermatozoa are separated into X- (female) and Y- (male) chromosome bearing populations based on their difference in DNA content. The resultant ‘sex-sorted’ spermatozoa are then able to be used in conjunction with other assisted reproductive technologies such as artificial insemination or in-vitro fertilization (IVF) to produce offspring of the desired sex. DNA damage in sperm cells may be detected by using Raman spectroscopy. It is not specific enough to detect individual traits, however. The sperm cells having least DNA damage may subsequently be injected into the egg cell by intracytoplasmic sperm injection (ICSI).

Sperm sorting utilizes the technique of flow cytometry to analyze and ‘sort’ spermatozoa. During the early to mid 1980s, Dr. Glenn Spaulding was the first to sort viable whole human and animal spermatozoa using a flow cytometer, and utilized the sorted motile rabbit sperm for artificial insemination. Subsequently, the first patent application disclosing the method to sort “two viable subpopulations enriched for x- or y- sperm” was filed in April 1987 and the patent included the discovery of haploid expression (sex-associated membrane proteins, or SAM proteins) and the development of monoclonal antibodies to those proteins. Additional applications and methods were added, including antibodies, from 1987 through 1997. At the time of the patent filing, both Lawrence Livermore National Laboratories and the USDA were only sorting fixed sperm nuclei, after the patent filing a new technique was utilized by the USDA where “sperm were briefly sonicated to remove tails”. USDA in conjunction with Lawrence Livermore National Laboratories, ‘Beltsfield Sperm Sexing Technology’ relies on the DNA difference between the X- and Y- chromosomes.

Prior to flow cytometric sorting, semen is labeled with a fluorescent dye called Hoechst 33342 which binds to the DNA of each spermatozoon. As the X chromosome is larger (i.e. has more DNA) than the Y chromosome, the “female” (X-chromosome bearing) spermatozoa will absorb a greater amount of dye than its male (Y-chromosome bearing) counterpart. As a consequence, when exposed to UV light during flow cytometry, X spermatozoa fluoresce brighter than Y- spermatozoa. As the spermatozoa pass through the flow cytometer in single file, each spermatozoon is encased by a single droplet of fluid and assigned an electric charge corresponding to its chromosome status (e.g. X-positive charge, Y-negative charge). The stream of X- and Y- droplets is then separated by means of electrostatic deflection and collected into separate collection tubes for subsequent processing.

While highly accurate, sperm sorting by flow cytometry will not produce two completely separate populations. That is to say, there will always be some “male” sperm among the “female” sperm and vice versa. The exact percentage purity of each population is dependent on the species being sorted and the ‘gates’ which the operator places around the total population visible to the machine. In general, the larger the DNA difference between the X and Y chromosome of a species, the easier it is to produce a highly pure population. In sheep and cattle, purities for each sex will usually remain above 90% depending on ‘gating’, while for humans these may be reduced to 90% and 70% for “female” and “male” spermatozoa, respectively. Some approaches to in vitro fertilization involve mixing sperm and egg in a test tube and letting nature take its course. But in about half of all infertility cases, a problem with the man’s sperm may require a more direct method. In these cases, a different process, called intracytoplasmic sperm injection (ICSI), in which a single sperm cell is injected directly into an egg, is sometimes used. With this one-shot opportunity, it’s important to choose a sperm cell with the best potential for success. A team at the University of Edinburgh, Scotland, has now announced a new technique to ensure that the best sperm win: analyzing their DNA for potential damage beforehand, and choosing those that are structurally sound.

To optimize success rates of IVF, selection of the most viable embryo(s) for transfer has always been essential, as embryos that are cryopreserved are thought to have a reduced chance of implanting after thawing. Recent developments challenge this concept. Evidence is accumulating that all embryos can now be cryopreserved and transferred in subsequent cycles without impairing pregnancy rates or maybe even with an improvement in pregnancy rates. In such a scenario no selection method will ever lead to improved live birth rates, as, by definition, the live birth rate per stimulated IVF cycle can never be improved when all embryos are serially transferred. In fact, selection could then only lower the live birth rate after IVF. The only parameter that could possibly be improved by embryo selection would be time to pregnancy, if embryos with the highest implantation potential are transferred first.

In the majority of human IVF cycles multiple embryos are created after ovarian hyperstimulation. The viability of these embryos, and as a consequence the chance for an embryo to successfully implant, is subject to biological variation. To achieve the best possible live birth rates after IVF while minimizing the risk for multiple pregnancy, one or two embryos that are considered to have the best chance of implanting are selected for transfer. Subsequently, supernumerary embryos with a good chance of implanting are selected for cryopreservation and possible transfer in the future while remaining embryos are discarded.

The best available method for embryo selection is morphological evaluation. On the basis of multiple morphological characteristics at one or several stages of preimplantation development, embryos are selected for transfer. However, with embryo selection based on morphological evaluation implantation rates in general do not exceed 35%, although varying results have been reported. This has resulted in a strong drive for finding alternative selection methods.

The best studied alternative selection method is preimplantation genetic screening (PGS). The classical form of PGS involves the biopsy at Day 3 of embryo development of a single cell of each of the embryos available in an IVF cycle and analysis of this cell by fluorescence in-situhybridization (FISH) for aneuploidies, for a limited number of chromosomes. Only embryos for which the analyzed blastomere is euploid for the chromosomes tested are transferred. Although this method of PGS has been increasingly used in the last decade, recent trials show that it actually decreases ongoing pregnancy rates compared with standard IVF with morphological selection of embryos.

In an effort to overcome some of the drawbacks of PGS using cleavage stage biopsy and FISH, new methods to determine the ploidy status of a single cell are developed, such as comparative genomic hybridization arrays or single nucleotide polymorphism arrays. Furthermore, in an attempt to avoid the confounding effects of chromosomal mosaicism, embryos are now biopsied at either the zygote or blastocyst stage. In addition, increasing time and money are invested in the development of high-tech, non-invasive methods to select the best embryo for transfer in IVF.

This Include metabolomic profiling, amino acid profiling, respiration-rate measurement and birefringence imaging.

In metabolomic profiling, spectrophotometric tests are used to measure metabolomic changes in the culture medium of embryos;
in proteomic profiling, proteins produced by the embryo and released into the culture medium are identified;
in amino acid profiling, amino acid depletion and production by the embryo is assessed using the culture medium;
in respiration-rate measurement, the respiration rate of embryos is assessed; and
in birefringence imaging, polarization light microscopy is used to assess the meiotic spindle or the zona pellucida.

Embryo donation (also known as embryo adoption) is the compassionate gifting of residual cryopreserved embryos by consenting parents to infertile recipients. At present, only a limited number of such transactions occur. In 2010, the last year for which U.S. data were available, fewer than 1000 embryo donations were recorded. These acts of giving, unencumbered by federal law, are being guided by a limited number of state laws. Moreover, the practice is sanctioned by professional societies, such as the American Society for Reproductive Medicine, subject to the provision that “the selling of embryos per se is ethically unacceptable.” As such, the not-for-profit donation of existing embryos by consenting parents comports with a triad of commonly held ethical attributes. First, donated embryos are not sold for profit. Second, donated embryos are (by original intent) generated for self-use. Third, donated embryos are the product of an unambiguous parental unit and as such are transferable. All told, embryo donation constitutes an established if limited component of present-day assisted reproduction.

Source References:

http://en.wikipedia.org/wiki/Sperm_sorting

http://www.technologyreview.com/news/411706/best-sperm-for-the-job/

http://humrep.oxfordjournals.org/content/26/5/964.long

http://www.nejm.org/doi/full/10.1056/NEJMsb1215894?query=genetics

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Genes involved in Male Fertility and Sperm-egg Binding

Posted in Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Chemical Genetics, Genome Biology, Genomic Testing: Methodology for Diagnosis, Human Immune System in Health and in Disease, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Population Health Management, Genetics & Pharmaceutical, Reproductive Biology & Bio Instrumentation, tagged DEFB126 protein, fertility, gene, genetics, In vitro fertilisation, infertility, male fertility, mutation, sperm, sperm-egg binding on October 8, 2012| 3 Comments »

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

An estimated 10 to 15% of couples suffer from infertility, and many treatment decisions rely on trial and error. A team of international scientists has found a common genetic variant which may explain why some men with normal sperm counts and good quality sperm are affected by infertility.

The study findings suggested that men with a variation in a gene which codes for a sperm-coating protein called beta defensin 126 (DEFB126) have a reduction in the protein coat on the outside of the sperm which makes it difficult for the sperm to ‘swim’ to the egg.

Dr Edward Hollox of the University of Leicester and co-author of the study said: ‘If you’ve got this gene variant you should allow that little bit longer if your partner’s planning to get pregnant’. The researchers, including scientists from the University of California and the Anhui Medical University in China, carried out the study on over 500 newly-wed Chinese couples who were trying for a baby.

They found that when men’s sperm lacked a coat of the DEFB126 protein, their wives were significantly less likely than expected to become pregnant.

Previous studies have shown that two copies of the genetic variant may be found in up to one quarter of men around the world, with about half of all men having one copy. The DEFB126 protein coat helps sperm to swim through cervical mucus and evade the woman’s immune system, as well as enabling it to attach to the walls of fallopian tubes.

The study showed, however, that men with two copies of the variant produced sperm that were less able to swim through a substitute to cervical mucus, hyaluronic acid gel. In macaques, it has already been shown that this protein is important in evading the immune system and the researchers believe the protein coat plays the same role in humans. Commenting on the study, Dr Allan Pacey, senior lecturer in Andrology at the University of Sheffield, said: ‘We actually understand very little about the subtle molecular events which occur in sperm as they make their journey through the woman’s body to fertilise an egg’. The research was published in the journal Science Translational Medicine. If replicated in future studies, these findings promise to guide choices about the timing and type of assisted reproduction interventions—and further hint at the possibility of treating sperm from del/del homozygotes to promote fertility.

A gene which helps sperm bind to an egg has been identified by scientists. Sperm-to-egg binding is an essential process during fertilization and although the preliminary studies were performed on mice, the gene may represent a new target for infertility treatments. Sperm from mice that had the gene switched off were only able to fertilise eggs from female mice three percent of the time compared to 80 percent fertilisation success in normal mice.

The gene codes for a protein called PDILT which helps another gene product to form and assemble correctly and then to reach the surface of a sperm. Once this happens the sperm is able to navigate the uterus and oviduct and penetrate the sticky outer layers of an egg. The study, which is published in PNAS (Proceedings of the National Academy of Sciences), also demonstrates the importance of cumulus cells, a cluster of cells that surround and protect the egg, as their presence allows sperm to bind to their target. Sperm from mice that had their PDILT gene switched off would not bind to a bare egg, but would bind to an egg surrounded by cumulus cells.

Co-author Dr Adam Benham from Durham University in the UK said that the PDILT protein is ‘an essential part of the navigation system of sperm. Like any navigation system, you have to programme where it is that you want to go and this protein plays an essential role in getting sperm to the right destination, in good shape, and in good time’. A question now for the scientists is whether the PDILT gene has as much importance in human fertility as it does in mice. ‘Mutations in the gene may be responsible for unexplained male fertility problems and further research may aid more effective IVF treatment‘, said Dr Benham.

Source References:

http://stm.sciencemag.org/content/3/92/92ps31.abstract

http://www.bionews.org.uk/page_142955.asp

http://www.itv.com/news/update/2012-05-01/scientists-discover-new-gene-key-to-fertility/

http://www.bionews.org.uk/page_102705.asp