CRISPR News – GenScript

Posted: June 28, 2018 at 5:41 am

CRISPR Plasmids

DNA plasmids for single guide RNA and/or Cas9 expression

Validated knock-out cell line service using CRISPR technology.

Genome-wide or pathway-specific CRISPR knock-out or activation libraries for screening experiments.

Validated knock-out and knock-in mutagenesis in bacteria and yeast.

CRISPR News

July 6, 2017

For the first time, researchers have been able to detect and characterize the mechanism of action by which the CRISPR complex binds and cleaves DNA using electron microscopy. Scientists at Harvard and Cornell have recently created near-atomic level resolution images of the CRISPR/Cas3 complex, a common CRISPR/Cas subtype, which provide structural data that can improve gene editing accuracy and efficiency.

To solve problems of specificity, we need to understand every step of CRISPR complex formation, states Maofu Liao, a co-author of the study and assistant professor at Harvard. Our study now shows the precise mechanism for how invading DNA is captured by CRISPR, from initial recognition of target DNA and through a process of conformational changes that make DNA accessible for final cleavage by Cas3.

This discovery uncovers a number of novel, overlapping mechanisms which prevent off-target site cleavage. In the CRISPR/Cas3 system, the assembled CRISPR complex first searches for a corresponding protospacer adjacent motif (PAM) sequence, which indicates a possible target site. Researchers discovered that as the CRISPR complex detects the PAM, it also bends DNA at a sharp angle, forcing a small portion to unwind. This allows an 11-nucleotide stretch of the CRISPR guide RNA to bind onto the target DNA, creating a seed bubble. The seed bubble acts as a fail-safe mechanism to check whether target DNA matches the guide RNA. If correctly matched, the bubble is enlarged and the remainder of the guide RNA binds onto the DNA forming an R-loop structure. Only once the full R-loop structure is formed does the Cas enzyme bind and cut the DNA in the non-target DNA strand. This study is the first to reveal the full sequence of events from seed bubble formation to R-loop formation.

Looking for an affordable and easy way to model disease in vivo?Interested in performing a genome-wide screen?Use CRISPR RNA/Cas9 Reagents or CRISPR Plasmids for high efficiency, customizable gene editing.

Xiao, Y. et al. Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System.Cell170, 48-60.e11 (2017).

June 28, 2017

Today, nearly 1 out of every 68 children born is diagnosed with autism spectrum disorder (ASD). Globally the disease is estimated to affect over 25 million people. And prevalence is expected to rise with ASD identifications doubling in the last decade.

ASD describes a variety of neurodevelopmental disorders which are often characterized by deficits in social communication and interaction, and restricted and repetitive behavior. While no specific causes for ASD have yet been found, a number of genetic and environmental risk factors have been identified. Most recently, a new study from Columbia Universitys Mailman School of Public Health, has discovered that prenatal fever increases autism risk by up to 40%.

Researchers monitored over 95,000 children born between 1999 and 2009. Of that population, 15,701 children were identified to have mothers reporting fever conditions during pregnancy. These children were found to have increased risk of ASD by 34%. Risk increase was highest, at 40%, when fever was reported during the second trimester. And ASD risk was increased by over 300% for the children of women reporting three or more fevers after the twelfth week of pregnancy.

Our results suggest a role for gestational maternal infection and innate immune responses to infection in the onset of at least some cases of autism spectrum disorder, states lead researcher, Associate Professor Mady Hornig. Additional studies are ongoing to determine the role of specific infectious agents in the development of ASD.

Hornig, M. et al. Prenatal fever and autism risk. Molecular Psychiatry (2017). doi:10.1038/mp.2017.119

June 22, 2017

Each year almost 200,000 people in the U.S. require emergency medical care for a serious allergic reaction. This number is expected to grow as food allergy incidence has increased by 50% in the last decade.

Allergies are caused from the hypersensitivity of the immune system to allergens in the environment. Recognition of these allergens triggers a T-cell-mediated immune response, producing cytokines which induce chronic inflammation and mucous hypersecretion.

In a recent study at the University of Queensland, Professor Ray Steptoe has been able to de-sensitize T-cells using a novel gene therapy treatment. Dr. Steptoes research team has engineered bone marrow stem cells to express transgenic allergen proteins. This effectively tricks the body into identifying the transgenic allergen as a self-antigen originating from within the body, leading to negative selection of any reacting T-cells. After treatment, the immune system is memory wiped alleviating airway inflammation and hyperreactivity.

Dr. Steptoe states that the eventual goal will be to devise a single-dose injectable therapeutic, which could replace the various short-term treatments that focus on alleviating allergy symptoms. Potential patents would be those individuals who are suffering from potentially lethal allergies or severe asthma.

AL-Kouba, J. et al. Allergen-encoding bone marrow transfer inactivates allergic T cell responses, alleviating airway inflammation. JCI Insight2, (2017).

June 15, 2017

Each year nearly 2 million people in the USA are infected by antibiotic-resistant bacteria. With antibiotic resistance on the rise, scientists have begun to turn to alternative antimicrobial treatments.

At the University of Wisconsin-Madison, scientists are developing a new probiotic “CRISPR pill” that is effective even against drug-resistant threats. Researchers from the lab of Jan-Peter Van Pijkeren have engineered bacteriophages expressing customized CRISPR guide RNA sequences. These CRISPR RNAs hijack the innate bacterial CRISPR immunity system present in infectious bacteria, causing them to self-destruct by creating lethal breaks in their own DNA. The bacteriophages are packaged in pill form in a mixture of probiotics, allowing them to survive the digestive tract until reaching the intestines.

By utilizing the innate immune system present in bacteria, the CRISPR pill bypasses the main mechanisms of antibiotic resistance. In addition, CRISPR pills may be superior to traditional antibiotics, because of their narrow targeting spectrum which can target specific bacterial species and strains. In contrast, broad-spectrum antibiotics kill off both “good” and “bad” bacteria. And overuse of traditional antibiotics has lead to the rising epidemic of antibiotic-resistant infections.

CRISPR and the CRISPR Associated system (Cas) is a powerful gene editing technology. Originally identified and characterized in bacteria, endogenous CRISPR systems act as an RNA-based defense mechanism against invading phage DNA.

CRISPR was adapted for genome editing in 2013 and has since been exploited for its ability to generate targeted double-stranded DNA breaks, which has revolutionized molecular biology protocols.1,2

This guide covers the basics of CRISPR experimental design and will prepare you to embark upon your own genome editing experiment.

Endogenous CRISPR systems fall into three categories type I, II and II. You can read more about these types in Makarova et al.3 Commercial CRISPR genome editing tools are adapted and simplified from endogenous type II systems and have the following components:

When gRNA and Cas9 are expressed together in a cell, a gRNA:Cas9 complex is recruited to the target DNA sequence, which is located immediately upstream of a motif called a protospacer adjacent motif (PAM).4 The PAM motif targeted by most commercial Cas9 enzymes is NGG (any nucleotide followed by two guanines).

Binding of the gRNA to target DNA occurs via complementary base-pairing between the genomic target sequence and the 20-nucleotide spacer on the gRNA. The Cas9 in the gRNA:Cas9 complex then cuts the genomic DNA, inducing a double-stranded break after the PAM sequence. Crucially, Cas9 cannot digest DNA unless bound to the gRNA, thus providing specificity to the system.

The editing process is completed by repairing the break using the endogenous Non-Homologous End Joining (NHEJ) pathway. While this DNA repair system is the most efficient repair pathway it is error prone, sometimes permitting small insertions or deletions, which can result in frameshifts and reduced protein production. An alternative option is to exploit the endogenous Homology Directed Repair (HDR) system by providing the HR template, as mentioned above. This is used when introducing targeted mutations.

Once you have designed and cloned the gRNA and HR templates, you cotransfect the Cas9 plasmid and your gRNA and HR donor vectors into the chosen cell line. Lipid transfection, electroporation or microinjection are all suitable transfection methods.

Optimizing recombination levels may take some trial and error. Choose a robust cell line (e.g., HEK) for troubleshooting. Once your experiment is up and running, you can move onto more expensive and less robust cell lines, if necessary.

Bear in mind that immortalized cell lines are not only cheaper than primary cells, but their recombination pathways are often less stringent. Therefore, you should ideally achieve a high level of recombination efficiency before moving to primary lines.

In the end, efficiency of your CRISPR experiment is part plan, part luck. The interaction between the system components and Cas9 is still not well understood. Fortunately, there are a few ways you can increase your odds:

If you have done everything right but are still experiencing low efficiency, then it is time to experiment. You may have better luck using sense and anti-sense templates. Others have reported better efficiency with asymmetrical arms.5 Be prepared to design a few setups the efficiencies of overlapping designs can vary widely and be ready to experiment to find the best design for your experiments. For more information about CRISPR, check out this free CRISPR handbook.

How to Optimize Your Lentiviral ExperimentsMarch 7, 2017

There are several aspects to consider if you want to optimize your lentiviral experiments. Check out these helpful tips before you embark on the incessant optimization experiments. Here are three common factors that may be affecting your viral titers:

The 293 cell line was derived from embryonic kidney cells and is commonly used for lentivirus production. HEK 293 cells are sensitive to passage number and should be replaced regularly; cells must be healthy and actively dividing. HEK 293Ts, which contain the SV40 T antigen, are more resilient and can be used for six months or longer with no significant reduction in virus titer.

Clumpy cell cultures with lots of senescent cells will not produce good titers. It is worth doing a test transfection on your cells before you try using them for virus production. If your transfection efficiency is low, then there is no point continuing with virus production, you will need to setup a new cell stock.

Remember:

There are a number different commercial and non-commercial transfection reagents available. Chemical reagents such as calcium phosphate and polyethylenimine (PEI) work effectively and are very budget-friendly. For transfection with PEI or a commercial lipid-based reagent, your 293 cells should be 90-95% confluent at the time of transfection.

Remember:

A lentivirus expression typically contains a transfer plasmid and a packaging plasmid. Plasmids are recommended to be cultivated from bacterial strains such as Stbl3, which have reduced frequencies of homologous recombination. Plasmids containing a Gateway cassette with the ccdBgene will require a compatible ccdB viable strain. Make sure after plasmid purification that plasmid quality is high and of a reasonable concentration (over 100ng/L).

When considering packaging plasmids, make sure not to confuse the second and third generation variants. Second generation transfer plasmids require the presence of HIV-1 Tat protein. Third generation transfer plasmids have eliminated Tat from the packaging system, but are still backwards compatible with second generation transfer plasmids.

Transfer plasmids are the most important factor in virus production, and can result in transduction efficiency differences of 10-50x. The length of sequence between the long terminal repeats can directly influence viral titer, and particle yield decreases as sequence length increases. Including multiple promoters within the transfer plasmid can also result in promoter interference, where the promoters adversely affect expression of the others, resulting in lower viral titer.

Fluorescence microscopy and flow cytometry are two methods that can be used to measure transduction efficiency. Remember though that protein expression can influence fluorescence, and weakly expressed proteins can lead to underestimated viral titer. Therefore, promoter should be a key consideration if transduction is assessed using these methods.

Remember:

Researchers uncover novel fat metabolism pathwayFebruary 20, 2017

A new study in Nature Communications discovered a neuropeptide hormone, FLP-7, which is capable of stimulating fat metabolism. This fat metabolism pathway is the first to be discovered which can activate fat burning without affecting food intake or movement.

FLP-7 had previously been identified over 80 years ago as a muscle stimulant, but no links to fat metabolism was ever established. Flashing forward to 2017, scientists at the Scripps Institute identified FLP-7 during a genetic screen as a suppressor of fat loss in C. elegans roundworms. By fluorescently tagging the hormone, researchers were able to track FLP-7 secretions from the brain in response to elevated serotonin levels. This FLP-7 could then be tracked through the circulatory system to the gut, where it activates fat burning.

Modifying serotonin levels results in serious side effects, broadly impacting food intake, movement and reproduction. Amazingly, adjusting levels of FLP-7 does not result in any obvious changes, worms continue to function normally, while just burning more fat. Researchers hope their finding spur additional research into the weight loss effects of FLP-7 mammalian homologs.

Palamiuc et al. A tachykinin-like neuroendocrine signalling axis couples central serotonin action and nutrient sensing with peripheral lipid metabolism.Nature Communications, 2017; 8: 14237 DOI:10.1038/ncomms14237

How does sugar effect health and aging?February 6, 2016

A new study in Cell Reports has linked sugar intake to lifespan. This process occurs through a newly discovered pathway in which sugar permanently reprograms gene expression, maintaining an altered state even if your diet has improved.

Using fruit flies as a model organism, researchers compared life span of flies consuming a 5% and 40% sugar diet. Any flies raised on the 40% sugar diet averaged a 7% shorter life span. Researchers discovered that excess sugar promotes insulin-signaling pathways which lead to the inactivation of FOXO. FOXO is a transcription factor which alters the expression levels of chromatin modifiers. Crucially, the reprogramming of these transcription networks could not be reversed upon a switch to the lower sugar diet. The study improves our understanding of how changes in diet and gene expression can affect the speed of aging.

Dobson et al.Nutritional Programming of Lifespan by FOXO Inhibition on Sugar-Rich Diets.Cell Reports, 2017; 18 (2): 299 DOI:10.1016/j.celrep.2016.12.029

How does vitamin C fight cancer?January 30, 2016

Vitamin C’s efficacy in cancer prevention has been hotly debated. But, new research has shown that direct, intravenous delivery of vitamin C can more than double survival rates of pancreatic cancer. By avoiding the digestive tract, scientists have been able to increase vitamin C levels in the blood by 100-500 times. And at these extreme concentrations, vitamin C is able to selectively kill cancer cells.

As Vitamin C breaks down through oxidation hydrogen peroxide is generated. Hydrogen peroxide is capable of forming free radicals which can be damaging to DNA. Interestingly, researchers discovered that tumor cells are much less efficient at removing hydrogen peroxide. Tumor cells were found to be deficient in catalase activity, the primary means of detoxifying hydrogen peroxide. On average, tumor cells were able to only metabolize hydrogen peroxide at half the rate of normal cells. And the addition of vitamin C to these tumor cells resulted in ATP depletion, DNA lesions, and cell growth reduced by more than 50%. Clinical trials pairing both high-dosage, intravenous vitamin C and chemotherapy are now underway and in Phase 2 testing.

Doskey et al.Tumor cells have decreased ability to metabolize H2O2: Implications for pharmacological ascorbate in cancer therapy.Redox Biology, 2016; 10: 274 DOI:10.1016/j.redox.2016.10.010

Postdoc vs Industry? Comparing the ReturnsJanuary 23, 2016

A new study published in Nature Biotechnology has found that biomedical postdoctoral opportunities provide diminishing returns in the labor market. Upon graduating, many aspiring postdocs will hope to land a career in tenure track academia, but only 20% of scientists ever manage to attain such a position. The impact from such a decision can be staggeringly high.

Taking a postdoctoral position can cost up to three years worth of lost salary over the first 15 years of a scientist’s career. In 2013, the median starting salary for postdocs in academia was $44,724, compared to $73,662 for postdocs in industry. The academic experience accrued does not improve salary potential either, as scientists switching to industry average salaries equivalent to new, entry-level employees. Overall, academics will average $12,002 lower than though who leave the field.

But current graduates should stay informed of their options, and measure the chance of landing a tenure-track position against the potential financial ramifications.

Kahn and Ginther, 2017. The impact of postdoctoral training on early careers in biomedicine.Nature Biotechnology 2017; 35 (1): 90 DOI: 10.1038/nbt.3766

New mechanism for cancer metastasis discoveredJanuary 16, 2016

Cell biologists at Mount Sinai have identified a combination of changes to oncogenic and tumor suppressor genes which allow for early dissemination of cancer cells before a primary tumor forms. These cells first migrate before attaining additional mutations which lead to uncontrolled cell proliferation. But, a majority of the disseminated cancer cells will remain quiescent. And due to their non-proliferative nature, these cells form a reservoir resistant to chemotherapy and other conventional cancer treatments.

This early dissemination is a result of the activation of the p38 and HER2 pathways. Pathway activation leads to a cell type transition from epithelial to mesenchymal cells, which promotes cell migration. This process occurs normally in development during the formation of mammary and pancreatic ducts. But, the over-activation of both pathways during oncogenesis instead allows cancer cells to migrate into the bloodstream and metastasize instead.

Harper et al., 2016. Mechanism of early dissemination and metastasis in Her2 mammary cancer.Nature DOI: 10.1038/nature20609

Hosseimi et al., 2016. Early dissemination seeds metastasis in breast cancer.Nature DOI: 10.1038/nature20785

Mechanism behind Zika microcephaly revealedJanuary 9, 2016

Zika infection during fetal development has been associated with microcephaly and other birth defects. New analysis of Zika viral proteins has identified the mechanism by which the virus damages brain cells.

Cell biologists at Boston Children’s Hospital have identified the viral enzyme NS3 as the main culprit in Zika-associated neural degeneration. NS3 functions in the cleavage and processing of other Zika viral proteins. But, NS3 also is capable of interacting with and damaging centrioles, which are required for spindle assembly and cell proliferation. These findings are corroborated by genetic studies which have identified an association between centriole stability and microcephaly.

NS3 may prove to be an important drug target for against Zika-related illnesses moving forward. NS3 inhibitors commonly used to protect against dengue, a related virus, were shown to be successful in preventing NS3 binding to centrioles.

Saey, Tina.Cell biologists learn how Zika kills brain cells, devise schemes to stop it ScienceNews ScienceNews, 13 Dec 2016

How Did Mammary Glands EvolveJanuary 2, 2016

Researchers have recently discovered a new network of genes and enhancers responsible for coordinating the formation of mammary glands. Interestingly, this regulatory network functions by hijacking existing limb development processes.

Hox genes are a subset of homeotic genes which control embryonic development and patterning. Hox genes have been shown to regulate limb, head, thoracic, abdomen, and mammary gland formation.

To better understand how some of these body structures evolved, geneticists at the University of Geneva and the Swiss Federal Institute of Technology in Lusanne screened for Hox gene activating sequences in the genome. One of the enhancer sequences identified, MBRE, was found to be responsible for activating Hoxd9, a gene required for mammary gland development. Interestingly, MBRE is conserved only in placental and marsupial mammals, and missing in egg laying mammals, such as the platypus.

But MBRE regulatory network is found to function in all tissues, indicating that the network was present prior to mammary gland evolution. The researchers propose that Hoxd gene regulation in mammary glands evolved by co-opting existing regulatory networks in other body structures.

CRISPR Gene Editing Tested in Humans for the First TimeDecember 12, 2016

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