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Archive for the ‘Gene Regulation’ Category


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

 

Researchers have embraced CRISPR gene-editing as a method for altering genomes, but some have reported that unwanted DNA changes may slip by undetected. The tool can cause large DNA deletions and rearrangements near its target site on the genome. Such alterations can confuse the interpretation of experimental results and could complicate efforts to design therapies based on CRISPR. The finding is in line with previous results from not only CRISPR but also other gene-editing systems.

 

CRISPR -Cas9 gene editing relies on the Cas9 enzyme to cut DNA at a particular target site. The cell then attempts to reseal this break using its DNA repair mechanisms. These mechanisms do not always work perfectly, and sometimes segments of DNA will be deleted or rearranged, or unrelated bits of DNA will become incorporated into the chromosome.

 

Researchers often use CRISPR to generate small deletions in the hope of knocking out a gene’s function. But when examining CRISPR edits, researchers found large deletions (often several thousand nucleotides) and complicated rearrangements of DNA sequences in which previously distant DNA sequences were stitched together. Many researchers use a method for amplifying short snippets of DNA to test whether their edits have been made properly. But this approach might miss larger deletions and rearrangements.

 

These deletions and rearrangements occur only with gene-editing techniques that rely on DNA cutting and not with some other types of CRISPR modifications that avoid cutting DNA. Such as a modified CRISPR system to switch one nucleotide for another without cutting DNA and other systems use inactivated Cas9 fused to other enzymes to turn genes on or off, or to target RNA. Overall, these unwanted edits are a problem that deserves more attention, but this should not stop anyone from using CRISPR. Only when people use it, they need to do a more thorough analysis about the outcome.

 

References:

 

https://www.nature.com/articles/d41586-018-05736-3?utm_source=briefing-dy

 

https://www.ncbi.nlm.nih.gov/pubmed/28561021

 

https://www.ncbi.nlm.nih.gov/pubmed/30010673

 

https://www.ncbi.nlm.nih.gov/pubmed/24651067

 

https://www.ncbi.nlm.nih.gov/pubmed/25398350

 

https://www.ncbi.nlm.nih.gov/pubmed/24838573

 

https://www.ncbi.nlm.nih.gov/pubmed/25200087

 

https://www.ncbi.nlm.nih.gov/pubmed/25757625

 

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

 

Long interspersed nuclear elements 1 (LINE1) is repeated half a million times in the human genome, making up nearly a fifth of the DNA in every cell. But nobody cared to study it and may be the reason to call it junk DNA. LINE1, like other transposons (or “jumping genes”), has the unusual ability to copy and insert itself in random places in the genome. Many other research groups uncovered possible roles in early mouse embryos and in brain cells. But nobody quite established a proper report about the functions of LINE1.

 

Geneticists gave attention to LINE1 when it was found to cause cancer or genetic disorders like hemophilia. But researchers at University of California at San Francisco suspected there was more characteristics of LINE1. They suspected that if it can be most harmless then it can be worst harmful also.

 

Many reports showed that LINE1 is especially active inside developing embryos, which suggests that the segment actually plays a key role in coordinating the development of cells in an embryo. Researchers at University of California at San Francisco figured out how to turn LINE1 off in mouse embryos by blocking LINE1 RNA. As a result the embryos got stuck in the two-cell stage, right after a fertilized egg has first split. Without LINE1, embryos essentially stopped developing.

 

The researchers thought that LINE1 RNA particles act as molecular “glue,” bringing together a suite of molecules that switch off the two-cell stage and kick it into the next phase of development. In particular, it turns off a gene called Dux, which is active in the two-cell stage.

 

LINE1’s ability to copy itself, however, seems to have nothing to do with its role in embryonic development. When LINE1 was blocked from inserting itself into the genome, the embryonic stem cells remained unaffected. It’s possible that cells in embryos have a way of making LINE1 RNA while also preventing its potentially harmful “jumping” around in the genome. But it’s unlikely that every one of the thousands of copies of LINE1 is actually being used to regulate embryonic development.

 

LINE1 is abundant in the genomes of almost all mammals. Other transposons, also once considered junk DNA, have turned out to have critical roles in development in human cells too. There are differences between mice and humans, so, the next obvious step is to study LINE1 in human cells, where it makes up 17 percent of the genome.

 

References:

 

https://www-theatlantic-com.cdn.ampproject.org/c/s/www.theatlantic.com/amp/article/563354/

 

https://www.ncbi.nlm.nih.gov/pubmed/29937225

 

https://www.nature.com/scitable/topicpage/transposons-the-jumping-genes-518

 

https://www.sciencedaily.com/releases/2018/06/180621141038.htm

 

https://www.ncbi.nlm.nih.gov/pubmed/16015595

 

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

 

The CRISPR-Cas9 system has proven to be a powerful tool for genome editing allowing for the precise modification of specific DNA sequences within a cell. Many efforts are currently underway to use the CRISPR-Cas9 system for the therapeutic correction of human genetic diseases. CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells.

 

CRISPR–Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR–Cas9.

 

Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells. Using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), an average insertion or deletion (indel) efficiency greater than 80% was achieved. This high efficiency of insertion or deletion generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs.

 

The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. These results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. As hPSCs can acquire P53 mutations, cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.

 

CRISPR-based editing of T cells to treat cancer, as scientists at the University of Pennsylvania are studying in a clinical trial, should also not have a p53 problem. Nor should any therapy developed with CRISPR base editing, which does not make the double-stranded breaks that trigger p53. But, there are pre-existing humoral and cell-mediated adaptive immune responses to Cas9 in humans, a factor which must be taken into account as the CRISPR-Cas9 system moves forward into clinical trials.

 

References:

 

https://techonomy.com/2018/06/new-cancer-concerns-shake-crispr-prognosis/

 

https://www.statnews.com/2018/06/11/crispr-hurdle-edited-cells-might-cause-cancer/

 

https://www.biorxiv.org/content/early/2017/07/26/168443

 

https://www.nature.com/articles/s41591-018-0049-z.epdf?referrer_access_token=s92jDP_yPBmDmi-USafzK9RgN0jAjWel9jnR3ZoTv0MRjuB3dEnTctGtoy16n3DDbmISsvbln9SCISHVDd73tdQRNS7LB8qBlX1vpbLE0nK_CwKThDGcf344KR6RAm9k3wZiwyu-Kb1f2Dl7pArs5yYSiSLSdgeH7gst7lOBEh9qIc6kDpsytWLHqX_tyggu&tracking_referrer=www.statnews.com

 

https://www.nature.com/articles/s41591-018-0050-6.epdf?referrer_access_token=2KJ0L-tmvjtQdzqlkVXWVNRgN0jAjWel9jnR3ZoTv0Phq6GCpDlJx7lIwhCzBRjHJv0mv4zO0wzJJCeuxJjzoUWLeemH8T4I3i61ftUBkYkETi6qnweELRYMj4v0kLk7naHF-ujuz4WUf75mXsIRJ3HH0kQGq1TNYg7tk3kamoelcgGp4M7UTiTmG8j0oog_&tracking_referrer=www.statnews.com

 

https://www.biorxiv.org/content/early/2018/01/05/243345

 

https://www.nature.com/articles/nmeth.4293.epdf

 

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

 

Biologists may have been building a more nuanced view of sex, but society has yet to catch up. True, more than half a century of activism from members of the lesbian, gay, bisexual and transgender community has softened social attitudes to sexual orientation and gender. Many societies are now comfortable with men and women crossing conventional societal boundaries in their choice of appearance, career and sexual partner. But when it comes to sex, there is still intense social pressure to conform to the binary model.

 

This pressure has meant that people born with clear DSDs (difference/disorder of sex development) often undergo surgery to ‘normalize’ their genitals. Such surgery is controversial because it is usually performed on babies, who are too young to consent, and risks assigning a sex at odds with the child’s ultimate gender identity — their sense of their own gender. Intersex advocacy groups have therefore argued that doctors and parents should at least wait until a child is old enough to communicate their gender identity, which typically manifests around the age of three, or old enough to decide whether they want surgery at all.

 

As many as 1 person in 100 has some form of “DSD” with or without external manifestation. Diagnoses of DSDs previously relied on hormone tests, anatomical inspections and imaging, followed by painstaking tests of one gene at a time. Now, advances in genetic techniques mean that teams can analyze multiple genes at once, aiming straight for a genetic diagnosis and making the process less stressful for families. Children with DSDs are treated by multidisciplinary teams that aim to tailor management and support to each individual and their family, but this usually involves raising a child as male or female even if no surgery is done.

 

The simple scenario that all learn is that two X chromosomes make someone female, and an X and a Y chromosome make someone male. These are simplistic ways of thinking about what is scientifically very complex. Anatomy, hormones, cells, and chromosomes (and also personal identity convictions) are actually not usually aligned with this binary classification.

 

More than 25 genes that affect sex development have now been identified, and they have a wide range of variations that affect people in subtle ways. Many differences aren’t even noticed until incidental medical encounters, such as a forty-six-year-old woman pregnant with her third child, found after amniocentesis that half her cells carry male chromosomes. Or a seventy-year-old father of three who learns during a hernia repair that he has a uterus.

 

Furthermore, scientists now understood that everyone’s body is made up of a patchwork of genetically distinct cells, some of which may have a different sex than the rest. This “mosaicism” can have effects ranging from undetectable to extraordinary, such as “identical” twins of different sexes. An extremely common instance of mosaicism comes from cells passing over the placental barrier during pregnancy. Men often carry female cells from their mothers, and women carry male cells from their sons. Research has shown that these cells remain present for decades, but what effects they have on disease and behavior is an essentially unstudied question.

 

References:

 

https://www.theguardian.com/science/2017/mar/02/cambridge-scientists-create-first-self-developing-embryo-from-stem-cells

 

https://www.ncbi.nlm.nih.gov/pubmed/25693544

 

http://onlinelibrary.wiley.com/doi/10.1002/ajmg.a.34123/abstract;jsessionid=A330AD995EE25C7A0AD5EA478694ADD8.f04t01

 

https://www.ncbi.nlm.nih.gov/pubmed/25091731

 

https://www.ncbi.nlm.nih.gov/pubmed/1695712

 

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

 

A mutated gene called RAS gives rise to a signalling protein Ral which is involved in tumour growth in the bladder. Many researchers tried and failed to target and stop this wayward gene. Signalling proteins such as Ral usually shift between active and inactive states.

 

So, researchers next tried to stop Ral to get into active state. In inacvtive state Ral exposes a pocket which gets closed when active. After five years, the researchers found a small molecule dubbed BQU57 that can wedge itself into the pocket to prevent Ral from closing and becoming active. Now, BQU57 has been licensed for further development.

 

Researchers have a growing genetic data on bladder cancer, some of which threaten to overturn the supposed causes of bladder cancer. Genetics has also allowed bladder cancer to be reclassified from two categories into five distinct subtypes, each with different characteristics and weak spots. All these advances bode well for drug development and for improved diagnosis and prognosis.

 

Among the groups studying the genetics of bladder cancer are two large international teams: Uromol (named for urology and molecular biology), which is based at Aarhus University Hospital in Denmark, and The Cancer Genome Atlas (TCGA), based at institutions in Texas and Boston. Each team tackled a different type of cancer, based on the traditional classification of whether or not a tumour has grown into the muscle wall of the bladder. Uromol worked on the more common, earlier form, non-muscle-invasive bladder cancer, whereas TCGA is looking at muscle-invasive bladder cancer, which has a lower survival rate.

 

The Uromol team sought to identify people whose non-invasive tumours might return after treatment, becoming invasive or even metastatic. Bladder cancer has a high risk of recurrence, so people whose non-invasive cancer has been treated need to be monitored for many years, undergoing cystoscopy every few months. They looked for predictive genetic footprints in the transcriptome of the cancer, which contains all of a cell’s RNA and can tell researchers which genes are turned on or off.

 

They found three subgroups with distinct basal and luminal features, as proposed by other groups, each with different clinical outcomes in early-stage bladder cancer. These features sort bladder cancer into genetic categories that can help predict whether the cancer will return. The researchers also identified mutations that are linked to tumour progression. Mutations in the so-called APOBEC genes, which code for enzymes that modify RNA or DNA molecules. This effect could lead to cancer and cause it to be aggressive.

 

The second major research group, TCGA, led by the National Cancer Institute and the National Human Genome Research Institute, that involves thousands of researchers across USA. The project has already mapped genomic changes in 33 cancer types, including breast, skin and lung cancers. The TCGA researchers, who study muscle-invasive bladder cancer, have looked at tumours that were already identified as fast-growing and invasive.

 

The work by Uromol, TCGA and other labs has provided a clearer view of the genetic landscape of early- and late-stage bladder cancer. There are five subtypes for the muscle-invasive form: luminal, luminal–papillary, luminal–infiltrated, basal–squamous, and neuronal, each of which is genetically distinct and might require different therapeutic approaches.

 

Bladder cancer has the third-highest mutation rate of any cancer, behind only lung cancer and melanoma. The TCGA team has confirmed Uromol research showing that most bladder-cancer mutations occur in the APOBEC genes. It is not yet clear why APOBEC mutations are so common in bladder cancer, but studies of the mutations have yielded one startling implication. The APOBEC enzyme causes mutations early during the development of bladder cancer, and independent of cigarette smoke or other known exposures.

 

The TCGA researchers found a subset of bladder-cancer patients, those with the greatest number of APOBEC mutations, had an extremely high five-year survival rate of about 75%. Other patients with fewer APOBEC mutations fared less well which is pretty surprising.

 

This detailed knowledge of bladder-cancer genetics may help to pinpoint the specific vulnerabilities of cancer cells in different people. Over the past decade, Broad Institute researchers have identified more than 760 genes that cancer needs to grow and survive. Their genetic map might take another ten years to finish, but it will list every genetic vulnerability that can be exploited. The goal of cancer precision medicine is to take the patient’s tumour and decode the genetics, so the clinician can make a decision based on that information.

 

References:

 

https://www.ncbi.nlm.nih.gov/pubmed/29117162

 

https://www.ncbi.nlm.nih.gov/pubmed/27321955

 

https://www.ncbi.nlm.nih.gov/pubmed/28583312

 

https://www.ncbi.nlm.nih.gov/pubmed/24476821

 

https://www.ncbi.nlm.nih.gov/pubmed/28988769

 

https://www.ncbi.nlm.nih.gov/pubmed/28753430

 

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

 

Scientists think excessive population growth is a cause of scarcity and environmental degradation. A male pill could reduce the number of unintended pregnancies, which accounts for 40 percent of all pregnancies worldwide.

 

But, big drug companies long ago dropped out of the search for a male contraceptive pill which is able to chemically intercept millions of sperm before they reach a woman’s egg. Right now the chemical burden for contraception relies solely on the female. There’s not much activity in the male contraception field because an effective solution is available on the female side.

 

Presently, male contraception means a condom or a vasectomy. But researchers from Center for Drug Discovery at Baylor College of Medicine, USA are renewing the search for a better option—an easy-to-take pill that’s safe, fast-acting, and reversible.

 

The scientists began with lists of genes active in the testes for sperm production and motility and then created knockout mice that lack those genes. Using the gene-editing technology called CRISPR, in collaboration with Japanese scientists, they have so far made more than 75 of these “knockout” mice.

 

They allowed these mice to mate with normal (wild type) female mice, and if their female partners don’t get pregnant after three to six months, it means the gene might be a target for a contraceptive. Out of 2300 genes that are particularly active in the testes of mice, the researchers have identified 30 genes whose deletion makes the male infertile. Next the scientists are planning a novel screening approach to test whether any of about two billion chemicals can disable these genes in a test tube. Promising chemicals could then be fed to male mice to see if they cause infertility.

 

Female birth control pills use hormones to inhibit a woman’s ovaries from releasing eggs. But hormones have side effects like weight gain, mood changes, and headaches. A trial of one male contraceptive hormone was stopped early in 2011 after one participant committed suicide and others reported depression. Moreover, some drug candidates have made animals permanently sterile which is not the goal of the research. The challenge is to prevent sperm being made without permanently sterilizing the individual.

 

As a better way to test drugs, Scientists at University of Georgia, USA are investigating yet another high-tech approach. They are turning human skin cells into stem cells that look and act like the spermatogonial cells in the testes. Testing drugs on such cells might provide more accurate leads than tests on mice.

 

The male pill would also have to start working quickly, a lot sooner than the female pill, which takes about a week to function. Scientists from University of Dundee, U.K. admitted that there are lots of challenges. Because, a women’s ovary usually release one mature egg each month, while a man makes millions of sperm every day. So, the male pill has to be made 100 percent effective and act instantaneously.

 

References:

 

https://www.technologyreview.com/s/603676/the-search-for-a-perfect-male-birth-control-pill/

 

https://futurism.com/videos/the-perfect-male-birth-control-pill-is-coming-soon/?utm_source=Digest&utm_campaign=c42fc7b9b6-EMAIL_CAMPAIGN_2017_03_20&utm_medium=email&utm_term=0_03cd0a26cd-c42fc7b9b6-246845533

 

http://www.telegraph.co.uk/women/sex/the-male-pill-is-coming—and-its-going-to-change-everything/

 

http://www.mensfitness.com/women/sex-tips/male-birth-control-pill-making

 

http://health.howstuffworks.com/sexual-health/contraception/male-bc-pill.htm

 

http://europe.newsweek.com/male-contraception-side-effects-study-pill-injection-518237?rm=eu

 

http://edition.cnn.com/2016/01/07/health/male-birth-control-pill/index.html

 

http://www.nhs.uk/Conditions/contraception-guide/Pages/male-pill.aspx

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Translation of whole human genome sequencing to clinical practice: The Joint Initiative for Metrology in Biology (JIMB) is a collaboration between the National Institute of Standards & Technology (NIST) and Stanford University.

Reporter: Aviva Lev-Ari, PhD, RN

 

JIMB’s mission is to advance the science of measuring biology (biometrology). JIMB is pursuing fundamental research, standards development, and the translation of products that support confidence in biological measurements and reliable reuse of materials and results. JIMB is particularly focused on measurements and technologies that impact, are related to, or enabled by ongoing advances in and associated with the reading and writing of DNA.

Stanford innovators and industry entrepreneurs have joined forces with the measurement experts from NIST to create a new engine powering the bioeconomy. It’s called JIMB — “Jim Bee” — the Joint Initiative for Metrology in Biology. JIMB unites people, platforms, and projects to underpin standards-based research and innovation in biometrology.

Genome in a Bottle
Authoritative Characterization of
Benchmark Human Genomes


The Genome in a Bottle Consortium is a public-private-academic consortium hosted by NIST to develop the technical infrastructure (reference standards, reference methods, and reference data) to enable translation of whole human genome sequencing to clinical practice. The priority of GIAB is authoritative characterization of human genomes for use in analytical validation and technology development, optimization, and demonstration. In 2015, NIST released the pilot genome Reference Material 8398, which is genomic DNA (NA12878) derived from a large batch of the Coriell cell line GM12878, characterized for high-confidence SNPs, indel, and homozygous reference regions (Zook, et al., Nature Biotechnology 2014).

There are four new GIAB reference materials available.  With the addition of these new reference materials (RMs) to a growing collection of “measuring sticks” for gene sequencing, we can now provide laboratories with even more capability to accurately “map” DNA for genetic testing, medical diagnoses and future customized drug therapies. The new tools feature sequenced genes from individuals in two genetically diverse groups, Asians and Ashkenazic Jews; a father-mother-child trio set from Ashkenazic Jews; and four microbes commonly used in research. For more information click here.  To purchase them, visit:

Data and analyses are publicly available (GIAB GitHub). A description of data generated by GIAB is published here. To standardize best practices for using GIAB genomes for benchmarking, we are working with the Global Alliance for Genomics and Health Benchmarking Team (benchmarking tools).

High-confidence small variant and homozygous reference calls are available for NA12878, the Ashkenazim trio, and the Chinese son with respect to GRCh37.  Preliminary high-confidence calls with respect to GRCh38 are also available for NA12878.   The latest version of these calls is under the latest directory for each genome on the GIAB FTP.

The consortium was initiated in a set of meetings in 2011 and 2012, and the consortium holds open, public workshops in January at Stanford University in Palo Alto, CA and in August/September at NIST in Gaithersburg, MD. Slides from workshops and conferences are available online. The consortium is open and welcomes new participants.

SOURCE

Stanford innovators and industry entrepreneurs have joined forces with the measurement experts from NIST to create a new engine powering the bioeconomy. It’s called JIMB — “Jim Bee” — the Joint Initiative for Metrology in Biology. JIMB unites people, platforms, and projects to underpin standards-based research and innovation in biometrology.

JIMB World Metrology Day Symposium

JIMB’s mission is to motivate standards-based measurement innovation to facilitate translation of basic science and technology development breakthroughs in genomics and synthetic biology.

By advancing biometrology, JIMB will push the boundaries of discovery science, accelerate technology development and dissemination, and generate reusable resources.

 SOURCE

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Other related articles published in this Open Access Online Scientific Journal include the following:

“Genome in a Bottle”: NIST’s new metrics for Clinical Human Genome Sequencing

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/09/06/genome-in-a-bottle-nists-new-metrics-for-clinical-human-genome-sequencing/

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