CRISPR Products and Services at GenScript
DNA plasmids for single guide RNA and/or Cas9 expression
|Cell Line Gene Editing
Validated knock-out cell line service using CRISPR technology.
Genome-wide or pathway-specific CRISPR knock-out or activation libraries for screening experiments.
|Microbial Gene Editing
Validated knock-out and knock-in mutagenesis in bacteria and yeast.
New Study Reveals CRISPR/Cas Mechanism of Action
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. Cell 170, 48-60.e11 (2017).
How Fever During Pregnancy Can Affect a Child’s Autism Risk
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 University’s 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
How an Immune System “Memory Wipe” Can Cure Allergic Reactions
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. Steptoe’s 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 Insight 2, (2017).
Can CRISPR Pills Replace Antibiotics?
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.
How to Design a CRISPR Experiment and Start Genome Editing
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.
The Basics of a Commercial CRISPR System:
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:
- Cas9: An endonuclease that induces a double-strand break in genomic DNA, allowing the removal of genes or DNA sequences, or the integration of foreign DNA at specific sites.
- Guide RNA (gRNA): This RNA has a scaffold sequence, which is needed for Cas9 binding, and a 20-nucleotide user-defined spacer or target DNA sequence.
- Homologous Recombination (HR) template (optional): This is a piece of DNA that contains the mutation(s) that you would like to introduce flanked by regions of homology.
How Does CRISPR Work?
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.
Target Sequence and Guide RNA Considerations:
- Before designing your gRNA, determine the exact sequence of your target DNA. Differences between your gRNA and target DNA can reduce efficiency. Therefore, check for any species- or cell-specific polymorphisms before you start.
- Next, identify all PAM sequences (NGG) within your region of interest. Once identified, you can hone in on the best potential target region.
- Design the gRNA and HR template (if using) based on the location of the PAM. The gRNA must match the target DNA. However, equally as important, the gRNA should not match any other “off target” genomic sites.
- Check libraries/Use online tools
- Before you start designing your own gRNA, make sure to check validated gRNA libraries such as GenScript's gRNA library.
- If you don't find what you need in existing libraries you can use a software tool, such as such as Desktop Genetics, GenScript, Geneious, Benchling or CRISPR Design (MIT) to aid your design. These tools can identify target sequences with PAM sequences, and rank the results by their specificity to the target region (i.e., their on- and off-target potential), potentially saving you a lot of hassle later.
- Size is an important consideration:
- For small changes of <50 bp, the cut site should be 10-30 nucleotides from the target region, and the HR template should have 50-80 nucleotide homology arms on each side.
- If you want to make larger changes (e.g., >100 bp), then the HR template requires longer homology arms (approximately 800 nucleotides long).
- After designing your templates clone them into donor vectors.
Transfection & Screening of CRISPR Constructs
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.
How to Increase your Chances of Successful CRISPR genome editing:
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:
- Check your HR template for Cas9/PAM sites. These sites can lead to template degradation.
- Screen for PAM sites and remove them by making silent mutations. And remember, NGG to NAG doesn't work, as NAG is a cryptic PAM site.
- Only use high quality intact DNA free of RNA and other contaminants. The market boasts a range of spin column-based kits for high quality DNA isolation.
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.
- Cong L, Ann Ran F, Cox D, Lin S, Barretto R, Habib N, et al. (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339:819-23. doi: 10.1126/science.1231143
- Mali P, Yang L, Esvelt K, Aach J, Guell M, DiCarlo J, et al. (2013). RNA-guided human genome engineering via Cas9. Science 339:823-6. doi: 10.1126/science.1232033.
- Markova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, et al. (2011) Evolution and Classification of the CRISPR-Cas systems. Nat Rev Microbiol 9:467-77. doi: 10.1038/nrmicro2577.
- Karvelis T, Gasiunas G, Siksnys 2. (2017) Methods for decoding Cas9 protospacer adjacent motif (PAM) sequences: a brief overview. Methods S1046-2023(16)30304-8. doi: 10.1016/j.ymeth.2017.03.006.
- Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE. (2016) Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol 34:339-44. doi: 10.1038/nbt.3481.
How to Optimize Your Lentiviral Experiments
March 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:
HEK 293 Cells
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.
- It is recommended to split your cells the day before transfection.
- Lentivirus particles are sensitive to pH, so adding HEPES buffer to the culture media can help to protect from pH extremes.
- If your plasmids contain an SV40 origin of replication you need to use 293T cells.
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.
- Different transfection reagents and incubation lengths may have different optimal levels for cell confluency.
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 ccdBccdB viable strain. Make sure after plasmid purification that plasmid quality is high and of a reasonable concentration (over 100ng/µL).
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.
- Use recA mutant bacterial strains to reduce mutation rates in your lentiviral plasmids.
- Check your DNA quality and plasmid sequence after purification.
- Record whether you are utilizing a second or third generation lentiviral packaging system.
- Remember that transgene length and promoter number can adversely affect your viral titer.
- Consider using fluorescence microscopy or flow cytometry to measure transduction efficiency in your control experiments.
Researchers uncover novel fat metabolism pathway
February 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.
How Does It Work?
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.
- Looking for an affordable and easy way to create a knock-out/in mutants? Use ready-for-transfection CRISPR RNA/Cas9 Reagents or custom CRISPR Plasmids for high efficiency, customizable gene editing.
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.
How does vitamin C fight cancer?
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.
- Looking for an affordable and easy way to create a knock-out/in mutants? Use ready-for-transfection CRISPR RNA/Cas9 Reagents or custom CRISPR Plasmids for high efficiency, customizable gene editing.
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 Returns
January 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.
- Alternative research staffing
- Increased postdoc pay
- Postdoc term limits
- Tenure-track opportunities for top new graduates
But current graduates should stay informed of their options, and measure the chance of landing a tenure-track position against the potential financial ramifications.
- Need to finish your thesis requirements? Looking for an affordable and easy way to create a knock-out mutants? Use ready-for-transfection CRISPR RNA/Cas9 Reagents or custom CRISPR Plasmids for high efficiency, customizable gene editing.
- Looking for career opportunities in the Biotech sector? Check out GenScript Careers.
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 discovered
January 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 revealed
January 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 Evolve
January 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 Time
December 12, 2016
Advances in CRISPR/Cas9 technology have enabled scientists to remove, add or modify genetic sequences in living organisms. But for the first time ever, CRISPR/Cas9 gene editing is being used in human cancer trials.
Researchers at Sichuan University in Chengdu, China are utilizing CRISPR modified cells to battle lung cancer as part of a preliminary clinical trial. Researchers have disabled the gene encoding for PD-1, a protein which normally serves to block the immune response, in white blood cells taken from patients. These activated immune cells can then be implanted back into the patient to target cancer cells.
The clinical trial will treat 10 patients, and mainly be used to gauge the safety of CRISPR/Cas9 therapeutic treatments. But the research community is following the results closely. Previous studies using antibodies to target PD-1 have had success in treating lung cancers, melanomas, and renal cancers.
CRISPR Demonstrates How Premature Aging Occurs
December 5, 2016
Premature aging diseases are rare genetic disorders often attributed to defects in DNA repair or telomere maintenance. In a recent, study scientists utilize CRISPR gene editing technology to introduce these defects in human cells.
Dyskeratosis congenita is an incurable premature aging disease, caused by a mutation in a protein, TPP1, present in stem cells. Using CRISPR/Cas9 technology, researchers have been able to recreate a gain-of-function TPP1 mutation into a human cell line to model the disease. Researchers demonstrated that the mutation is sufficient to affect telomere maintenance in cells, resulting in shortened telomeres. Researchers believe that in the future, gene editing with CRISPR/Cas9 will be able to cure the cellular symptoms of dyskeratosis congenita.
- Conducting gene deletion or insertion experiments of your own? Use ready-for-transfection CRISPR RNA/Cas9 Protein for high efficiency, customizable gene editing.
CRISPR Reveals How Snakes Lost Their Limbs
November 28, 2016
Limbs disappeared in snake ancestors over 100 million years ago. Today, most snakes lack any remnants of limbs, with the exception of pythons and boas, which maintain vestigial leg spurs.
While comparing genomes from boas, pythons, and more advanced snakes, researchers have recently identified changes to an enhancer element known as ZRS. The ZRS enhancer is a long-range regulator of Sonic hedgehog (Shh), a gene involved in a number of developmental processes, including limb development.
In order to determine the importance of these changes, researchers utilized CRISPR/Cas9 technology in mice replacing the normal mouse ZRS sequence with the ZRS sequences from other animals. Replacing the mouse ZRS sequence with human or even fish homologous sequences had no effect on normal limb development. But mutant mice containing the python ZRS sequence became "serpentized" and display extremely stunted limb growth, indicating the importance of ZRS in snake limb evolution.
Competition in Mice Drives Reproductive Arms Race
November 21, 2016
While examining mating strategies in mice, researchers have identified a gene, PrKar1a, which makes sperm faster and more competitive. Interestingly, changes in PrKar1a influence not only sperm fitness, but mating behavior as well.
Oldfield mice and deer mice are two closely related species with very different mating habits. While the oldfield mouse is strictly monogamous, the deer mouse is promiscuous and often rears litters from more than one mate. The reason is due to a genetic variant of PrKar1a present in deer mice.
The deer mouse PrKar1a variant gives rise to a larger sperm midpiece, which house mitochrondria to power locomotion of the sperm tail. Researchers believe that the increasing competition has selected for the evolution of PrKar1a, faster sperm, and promiscuous behaviors. Interestingly, defects in PrKar1a has also been associated with similar defects for male fertility in humans.
- Conducting gene deletion or insertion experiments of your own? Use ready-for-transfection CRISPR RNA/Cas9 Protein for high efficiency, customizable gene editing.
How Does Lithium Restores Neuron Synaptic Function?
November 14, 2016
Lithium mood stabilizers have been used to treat psychiatric disorders for decades. But the mechanism of action for lithium remained unknown until recently. New research on the Wnt signaling pathway has revealed how lithium restores neuronal function.
The Wnt signaling pathway is required for the development of the early brain and late neural differentiation processes. Abnormal Wnt signaling has been linked to neurological diseases, including schizophrenia and bipolar disorder. Researchers have shown that loss-of-function Wnt mutations result in a reduction of dendritic spines, neural projections which form synapses between different neurons.
Interestingly, lithium treatment in Wnt pathway mutant mice restored both the number of neuronal dendritic spines and normal behavior. Lithium blocks GSK-3β, an enzymatic inhibitor of the Wnt pathway. By disabling this inhibitor, lithium partially restores Wnt function, rescuing synapse formation in neurons.
Sex Determination by Horizontal Gene Transfer
November 7, 2016
For most animals, sex is determined through the presence or number of sex chromosomes, but new research studies are demonstrating that sex determination can be much more complicated.
In the pill bug Armadillidium vulgare (female heterogamety), endogenous sex determination genes have actually degenerated from disuse. Instead, pill bugs are "feminized" by the presence of Wolbachia bacteria which infect the testes and ovaries of these insects. But interestingly, uninfected pill bug populations are still able to maintain female-to-male sex ratios.
How to get female pill bugs without endogenous sex determination or Wolbachia bacteria?
Genetic analysis has traced the genetic feminizing factor to Wolbachia bacterial genes which have previously been inserted into the pill bug genome through horizontal gene transfer. Gene transfer is made possible due to bacteriophages harbored in Wolbachia, and has effectively created a new functional sex chromosome in these pill bugs.
Dog Genes Provide Clues to Understanding Autism
October 31, 2016
In a recent study, scientists uncovered the genetic underpinnings of dog sociability. By using a series of puzzles, researchers were able to quantify the willingness of beagles to seek help from nearby humans. When scanning the DNA of the beagles, researchers discovered genetic differences between the dogs that spent the least and most time in close, physical contact with the people participating in the study. The researchers suggest that there are genetic variants which make dogs more sociable, and that these variants have been selected for during domestication.
From this study, five genes were identified and linked to dog social behavior. Interestingly, four of these genes had human homologs which were associated with behavioral disorders in humans, including autism spectrum disorder, schizophrenia and attention deficit hyperactivity disorder. The results suggest that similar mechanisms may be used to regulate behavior in both humans and dogs. The researchers believe that dogs may prove to be a valuable tool to study human behavioral disorders in the future.
Use CRISPR/Cas9 technology to further your own studies. CRISPR can be used to create knock-out and knock-in mutations in under 8 weeks.
Retinal Cone Cells Do More Than Just Sense Color
October 24, 2016
Conventional wisdom identifies two types of light-sensing cells in the eye, rods and cones. Rod cells are used for peripheral and low-light vision. Cone cells sense color, identifying three different types of shades, greens, reds, and blues. But recent research shows that not all of these cones actually sense color.
By shining directed light onto cone cells and measuring the type, proportion, and repeatability of the sensations produced, researchers were able to identify two distinct signals. While 1/3rd of cone cells released a chromatic signal to the brain, 2/3rd of cone cells released an achromatic signal. This achromatic signal corresponds to higher resolution details, such as edges and lines that are important for vision.
Earlier this year, researchers have utilized CRISPR technology to replace a genetic mutation responsible for retinitis pigmentosa, an inherited condition responsible for more than 1.5 million cases of blindness worldwide. This marks the first time researchers have successfully repaired a defect gene in stem cells derived from patients.
CRISPR Genome Editing to Save Endangered Species
October 17, 2016
Human development has triggered a biodiversity crisis, with hundreds to thousands of species going extinct each year. Scientists have proposed a new solution for one of the leading causes, invasive species, by using a new genome editing strategy known as the gene drive.
Gene drives use CRISPR/Cas9 technology to overwrite the laws of inheritance, increasing the chances of trait inheritance in offspring from 50% up to 100%. First, a CRISPR/Cas9 cassette is designed to target a gene sequence essential for fertility. Next, the cassette is inserted into the target sequence, creating a heterozygous mutant. Expression of CRISPR/Cas9 will result in a double-stranded break in the wild-type chromosome pair, and homology-dependent repair will utilize the CRISPR/Cas9 cassette as a donor template, resulting in a sterile homozygous mutant. To reverse the gene drive effects, a new cassette containing a wild-type donor template can be used. If utilized properly, gene drives can remove invasive species and predators, such as rats or mosquitos, within generations.
Fighting sickle cell disease with CRISPR modified fetal hemoglobin
October 13, 2016
A new study in PNAS has identified a novel approach utilizing fetal hemoglobin to treat sickle cell disease. Hereditary persistence of fetal hemoglobin (HPFH) is a benign condition, where fetal hemoglobin production persists into adulthood. HPFH has been found to be beneficial in individuals with sickle cell disease (SCD), because fetal hemoglobin is capable of blocking hemoglobin polymerization, attenuating SCD symptoms.
Using CRISPR/Cas9 genome editing, researchers have mimicked the naturally occurring Sicilian HPFH mutation in hematopoietic stem cells. These stem cells differentiated into viable erythrocytes expressing fetal hemoglobin. These stem cells can then be transplanted back into patients as a therapeutic treatment, reducing the severity of SCD symptoms. This technique demonstrates a new transplantation strategy using CRISPR/Cas9-modified cells to treat medical conditions in humans, and paves the way for patient testing in the near future.
Researchers harvest CRISPR-modified crops for consumption
October 3rd, 2016
For the first time ever, researchers at Umeå University, Sweden have harvested CRISPR-modified plants for consumption. On August 16th, genetically modified cabbage was prepared for a historic pasta meal by plant scientist Stefan Jannson and radio host Gustaf Klarin. These cabbage plants had previously been edited to remove the gene encoding PsbS, a photosystem II protein which acts a release valve for excess absorbed light.
How does this change current GMO (genetically modified organism) regulations?
Recent rulings this year by European and American authorities have exempted certain CRISPR/Cas9-edited crops from GMO legislation. Authorities on both continents have determined that crops will only be classified as GMOs if they contain inserted foreign DNA. Under this interpretation, CRISPR-edited crops with deleted endogenous DNA sequences can be grown without government oversight or permission. This ruling cuts the regulatory tape on CRISPR/Cas9 technology, creating new research and commercial opportunities in agriculture.
Hands and fins share same evolutionary connection
September 26, 2016
The evolution of tetrapod limbs and fins in fish has long been of interest to evolutionary biologists. In a recent Nature Letter, scientists learned more information on not only how limbs and fins develop, but also the genetic link. Using CRISPR/Cas9 and fate mapping strategies, the authors discovered that development of both fins in fish and digits in mice are controlled by the same gene: Hoxa13. What does this commonality reveal about our evolutionary history?
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Scientists engineer bacteria with a 57-codon genome
September 19, 2016
Recoding the microbial genome has the potential to confer new biological functions to bacteria. These genetically recoded organisms, or "GROs," could then be used to enable biocontainment or confer resistance to infection by viruses. In a recent Science report, George Church's group set out to create this new GRO by creating an engineered bacterium with a 57-codon genome.
How were these GROs designed and assembled?
The team approached this whole-genome codon replacement by first identifying which codons could be replaced. Once identified, the scientists computed the recoded genome, which required over 148,000 total changes. Using both gene synthesis and CRISPR/Cas9, they were able to construct and parse together the fragments. While the recoded genome has not fully been synthesized, this project demonstrates how close we are to the creation of synthetic organisms.
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Exercise prolongs lifespan by enhancing telomere transcription
September 12, 2016
Alterations in telomerase activity, and consequently telomere integrity, contribute to normal cellular aging. However, it has been unknown if counteracting this decline in integrity could promote longevity. In a recent report published in Science Advances, scientists report that increased metabolism, as a result of exercise, can actually directly influence telomere fitness.
How does exercise protect against aging?
Recently, it has been discovered that telomeric repeat-containing RNA (TERRA) molecules associate with telomeres and potentially have a protective role. In this study, the authors found that TERRA levels can actually increase in individuals after exercise. In turn, the TERRA appear to function as ROS scavengers, further protecting the telomeres from any damage. Further study will be required to determine if TERRA could truly be a therapeutic target for protection against aging.
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Junk DNA controls backbone length by regulating Oct4 expression
September 5, 2016
What controls the number of ribs an organism has, and what separates us from a snake? This is a question that was recently investigated and reported in Developmental Cell. Interestingly, the authors were able to track trunk length development to the pluripotency gene Oct4. Regulation of Oct4 expression contributed to the difference in trunk length observed in mice and in snakes, and upon further study, regulation of Oct4 expression was controlled by a surprising source. Oct4 expression was being controlled upstream of the Oct4 locus – specifically a 250bp fragment – which when overexpressed could increase rib count in mouse embryos – similar to a snake.
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Cancerous and healthy breast tissue confirmed to have distinct bacterial profiles
August 29, 2016
The role our microbiome plays in maintaining our health has recently become quite clear. However, while there has been speculation that the microbiome plays a role in breast cancer carcinogenesis, the distinct microbial populations have not been easy to analyze using non-sterile techniques. There have been two recent reports now that suggest in fact there is a distinct difference between microbial populations not only between skin and breast tissue, but between healthy and diseased tissue. Under sterile conditions, in a recent publication in Scientific Reports, the authors found that malignant tissue was enriched with bacterial taxa normally found in lower abundance in healthy tissue. These species included Fusobacterium, Atopobium, Gluconacetobacter, Hydrogenophaga and Lactobacillus. Further study will be required to fully understand how this information can help prevent breast cancer.
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Synthetic bacteria engineered to destroy tumors by releasing anti-tumor toxins
August 22, 2016
Can bacteria be engineered to fight cancer? While this principle is not novel, the results of a study recently published in Nature suggests we are a step closer. In this report, the authors engineered a synthetic circuit, consisting of an activator, a hemolysis gene, and a lysis gene. E.coli were then transformed with these circuits and assessed in vitro. The authors then delivered the modified cells into tumors, where following induction lysed and released the anti-tumor toxin, haemolysin E, into mouse tumors. This treatment method was effective in killing the surrounding tumor cells without risk of bacterial infection. In addition, in combination with the chemotherapy drug 5-FU, the modified bacteria deterred malignant tumor growth in mouse models.
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Inhibiting TRIP8b gene expression relieves depression-like symptoms in mice
August 15, 2016
Depression has serious physical and emotional impacts on many individuals. In a recent Molecular Psychiatry report, a new gene target has been revealed that when inhibited, produces antidepressant effects. Specifically, knock-out of the TRIP8b gene in the hippocampus reduces depressive behavior in mouse models.
How can this finding accelerate gene therapy for the treatment of depression?
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Obesity and food preferences have been linked to transgenerational epigenetics
August 8, 2016
There have been multiple indications that genetics plays a major role in obesity; however, a recent report in Molecular Metabolism suggests that this role goes back more generations than previously thought. In this study, multigenerational metabolism and food preferences were compared in F0, F1, F2 and F3 lean and obese mouse models. They observed that progeny of F0 generations, or "grandparents", that had a propensity to overeat and gain weight, also had a preference for gaining weight when exposed to a junk diet. Interesting, this trend was only observed in males, and suggests that small amounts of RNA in sperm might be involved.
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Additional evidence of contagious cancer has been identified in bivalve invertebrates
August 1, 2016
While cancer is predominantly spread via oncogenic mutations to somatic cell genomes, there are an increasing number of cases of cancer transmission in mammals and invertebrates. While originally these cases were presumed to be few, a recent publication in Nature has revealed even more examples. In this report, the authors observed cross-species transmission of disseminated neoplasia, a leukemia-like disease in a variety of bivalve species.
In this study, the authors collected mussels (M. trossulus) from different regions in Canada and analyzed the genotypes of neoplastic cells. Interestingly, the genotype of these cancerous cells did not match the host cell genotype, suggesting they originated from another species. Similar situations were observed in cockles (C. edule), and clams (P. aureus). Together, their results suggest that transmissible cancer may be more of a threat than previously thought.
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Functional pituitary tissue created from human pluripotent stem cells
July 25, 2016
Hypopituitarism, a disease which impacts proper pituitary gland function, is difficult to treat and is the cause of many age-related and growth defects. While hormone-replacement therapies remains the most viable treatment option, quality of life is significantly compromised, and the treatment is very expensive. Stem cells continue to have the most potential as a pituitary cell replacement; however, successful differentiation into functional, hormone producing cells is an on-going challenge.
To address this, a group recently published in Stem Cell Reports a streamlined method to direct differentiation of human PSCs into pituitary cells. Using a 3D organoid culture method, PSCs were progressively exposed to growth factors, specifically FGF8 and BMP-2, to control their differentiation and tailor which hormonal cell types they became. Additionally, when these cells were transplanted into rats with hypopituitarism, normal hormone release resumed.
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CRISPR and RNAi screens reveal new therapeutic targets against flavivirus infection
July 18, 2016
Human flavivirus infections, including the Zika and Dengue virus, have become significant global health concerns in recent years. To accelerate the development of effective therapeutics against these viruses, a group recently reported in Cell a comprehensive RNAi and CRISPR screen to identify essential genes for viral replication. Which genes were identified and what will these findings mean in the fight against Zika?
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Thiosulfate transferase (TST) identified as a new therapeutic target for type 2 diabetes
July 11, 2016
Obesity continues to be prevalent in society, and individuals with diabetes are particularly predisposed to weight gain. In an effort to identify novel therapeutic targets for this disease, a recent publication in Nature Medicine has uncovered a new genetic target, thiosulfate transferase (TST) that can protect against diet-induced obesity and diabetes in mouse models. What is the mechanism behind how TST improves insulin sensitivity?
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Phages evolved to protect themselves from bacterial immune systems with CRISPR antidotes
July 5, 2016
CRISPR, a component of the adaptive immune system in bacteria, is one of the primary systems developed to evade bacteriophage infection. Yet like many species, persistent survival is the goal, and it turns out that bacteriophages have also found away around this immune response. A recent report in Nature Microbiology has revealed that bacteriophages have developed anti-CRISPR proteins that inactivate the CRISPR/Cas9-mediated immune response.
What are these CRISPR antidotes?
From the Pseudomonas phage genome, the authors found nine families of proteins that appeared to inhibit CRISPR function. These proteins were found across multiple species, and target I-E and I-F CRISPR systems. The anti-CRISPR genes that encode these proteins, or aca1, promote bacteriophage survival and replication following infection. Together these results demonstrate how phages have evolved to override this intricate immune defense.
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New CRISPR update: CRISPR c2c2 enzyme programmed to specifically target RNA
June 27, 2016
CRISPR/Cas9 has now become a very well established technology for creating highly specific edits to DNA. Among the CRISPR systems that have been identified, one has been of particular interest due to the presence of its Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, which have known RNase activity. Given this, Feng Zhang's group hypothesized that this protein, known as C2c2, may also have RNA-targeting functions that could be leveraged for RNA-editing.
What is the function of C2c2?
In the recent Science report, the authors observed that C2c2 could be engineered to target ssRNA via a 28nt crRNA (compared to the 20nt crRNA sequence required for DNA-specific editing). By changing basic residues in the HEPN domains to neutral, alanine residues, C2c2 could be inactivated to function as an RNA-targeting protein as opposed to a nuclease. Ultimately this finding expands the potential applications of CRISPR to both the precise editing and modification of ssRNA.
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CRISPR-based DNA barcoding method accelerates whole organism-lineage tracing
June 20, 2016
Mapping the origin of cells over the course of embryogenesis and differentiation has historically been a complex task, generally due to a lack of adequate technologies for cell tracing. A study recently published in Science, however, has attempted to address this issue using none other that the CRISPR/Cas9 gene editing technology. Considering the precision of CRISPR/Cas9, the authors hypothesized that they could use CRISPR to create unique patterns of mutations in non-coding parts of the genome, which would be passed on from cell to cell as they divide and differentiate. Thus by tracking the mutation patterns within this "DNA barcode," they could identify at a granular level the origin of adult, differentiated cells.
In this study, the authors delivered gRNA/Cas9 vectors to cells at different stages of embryogenesis to create the unique mutation patterns in the cell genome. As a result, diverse combinations of alleles that were retained as cells divided. Through this they reconstructed lineage relationships of different organs in zebrafish models, confirming a new and effective new method for tracing adult cells to their precursors.
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CRISPRainbow: a new imaging method that labels up to 7 chromosomal loci simultaneously
May 23, 2016
Tracking live DNA within cells has gotten a little easier – and more colorful. In a recent Nature Biotechnology paper, researchers out of UMass Medical School have engineered CRISPR/Cas to target and illuminate specific locations within the genome. Fluorescent proteins, including BFP, RFP and GFP, were fused to guide RNAs (gRNA), and co-expressed with an inactive Cas9 protein (dCas9) to illuminate specific chromosomal sites. Up to 7 different colors could be generated by combining the three fluorescent molecules on stem loops of the gRNA sequence. Not only can this CRISPR-based method detect more locations, but the ease at which this technology can be implemented will revolutionize live cell imaging.
A new CRISPR-based system facilitates trait mapping via targeted recombination events
May 16, 2016
While linkage and association studies are the most effective methods to link genes to specific phenotypic traits, identifying the exact chromosomal location can be almost impossible. Yet a recent Science report may have found a solution with one of today's hottest technologies – CRISPR/Cas9. Since homologous recombination events are generally rare, scientists took advantage of the CRISPR/Cas9 system to produce targeted recombination events at higher frequency in mitotic cells.
What's the advantage of this CRISPR-based system?
In this report, gRNAs were designed to target specific loci on chromosomes to generate loss of heterozygosity (LOH). As a proof of concept, the authors used CRISPR to fine-map manganese sensitivity in S. cerevisiae. The resolution of CRISPR enabled them to identify the gene variants, specifically Pmr1 variants, that are responsible for mediating the trait phenotype.
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CRISPR/Cas9 used to remove entire HIV-1 genome from infected T-cells
May 9, 2016
CRISPR/Cas9 has therapeutic potential for many diseases, and this has recently been confirmed in a Scientific Reports publication. In this study, the authors successfully adapted CRISPR to eliminate the entire HIV-1 genome from T cells in vitro. To accomplish this, gRNAs were designed to target the HIV-1 provirus, between the 5' and 3' LTRs, in infected CD4+ T cells. Once the provirus was effectively removed from the cell, they demonstrated that viral replication was eliminated completely, without affecting viability and activity of the T cell. So for the first time, the feasibility of CRISPR as a therapeutic for controlling AIDS may finally become a reality.
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Scientists use CRISPR/Cas9 to track RNA translocation in living cells
April 25, 2016
There are a variety of diseases that are in part caused by dysfunctional trafficking of RNA from the nucleus for translation. To enable RNA tracking within a live cell, and to get more insight into how trafficking dysfunction accelerates disease, a group out of UCSD has turned to the revolutionary gene editing tool, CRISPR/Cas9. To difference is though, that the group altered the Cas9 protein to specifically target RNA as opposed to DNA.
How can CRISPR track RNA in living cells?
In this report published in Cell, the RNA-targeting Cas9 (RCas9) recognizes an RNA-specific PAM sequence (PAMmer) to enable localization to RNA. With GFP fused to the RCas9, the authors could visualize GAPDH, ACTB, TFRC and CCNA2 mRNA movement within the cell.
Adaptations to the CRISPR/Cas9 technology enable production of biofuel precursors in yeast
March 7, 2016
Yeast, among other microbes, are powerhouses in metabolic engineering because they are readily adaptable to produce chemicals and other compounds for biofuels, food, and other industrial products. The challenge, however, has been to optimize gene editing techniques in order to produce these compounds. While there have been many studies detailing the potential of CRISPR/Cas9 gene editing in mammalian cells, this technology has been more difficult to implement in yeast. In a recent ACS Synthetic Biology paper, the authors tackled this challenge by adapting the CRISPR/Cas9 system for use in the yeast strain Yarrowia lipolytica.
How was the CRISPR system optimized?
In this study, the authors aimed to use Y. lipolytica to convert sugars to lipids and hydrocarbons. They found that editing in yeast was improved using a synthetic RNA Polymerase III promoter, SNR52, to drive expression of guide RNA. With this system, they could target genes involved in peroxisome biogenesis, ultimately enabling CRISPR/Cas9 genetic engineering in the strain.
Click here to learn more about how GenScript's microbial genome engineering services can accelerate the field of metabolic engineering.
CRISPR/Cas9 gene editing restores dystrophin gene expression to alleviate muscular dystrophy in mice
January 25, 2016
Duchenne muscular dystrophy (DMD) is a deadly disease affecting 1 in 3500 boys, resulting from a mutation in the dystrophin (Dmd) gene that leads to compromised integrity of muscle cell membranes. While traditional gene therapy to repair this mutation is ineffective because its size, a recent report in Science presents new hope with the help of CRISPR genome editing.
How might CRISPR/Cas9 reverse the effects of DMD?
In this study, the authors hypothesized that CRISPR could be used as an exon-skipping approach to remove the faulty exon, exon 23, that is responsible for DMD. AAV vectors were used to deliver guide RNA targeting exon 23 along with the Cas9 endonuclease into Dmd mutant mice. After 3 weeks, they observed restoration of dystrophin expression and muscle function in mice.
Beyond gene editing: CRISPR/Cas9 is successfully used to identify functional enhancers in the non-coding genome
January 22, 2016
Alterations to enhancer regions can directly influence cell growth, and so identifying these regions can accelerate development of cancer therapeutics. Since there are hundreds of thousands of enhancers in the genome, screening for these regions, such as by reporter assays, has been a serious undertaking. However, in a new study published in Nature Biotechnology, a genome-wide screening procedure has been developed to quickly screen for important regions of the non-coding genome that are implicated in tumorigenesis pathways.
How can CRISPR find enhancer elements?
In this study, a CRISPR library was prepared with over 1,000 gRNA vectors that target genomic sites bound by the transcription factors p53 and ERα – both of which control cell proliferation and are implicated in cancer progression. The libraries were expressed in human BJ cells which contain inducible HRAS (BJ-RASG12V) and from the screen the authors found three enhancer elements that were important for p53 function, as well as another three enhancer elements important for ERα function. Together, these results validate how powerful the CRISPR technology can be for unraveling the functions of the non-coding genome.
Learn more about CRISPR gene editing services at GenScript.
Re-engineered Cas9 endonuclease has improved specificity
January 5, 2016
CRISPR/Cas9 has quickly revolutionized the realm of genome editing for its ease of use and efficacy. The technology employs an endonuclease, Cas9, which creates targeted double strand breaks through the guidance of sequence-specific guide RNAs. While one of the most accurate technologies available, there is still a chance that Cas9 will bind to and cut non-target strands. To address this, Feng Zhang's group at the Broad Institute engineered an even more specific Cas9 enzyme.
How was Cas9 design improved?
In the report published in Science, Zheng's group observed that off-target binding was the result of interactions between positively charged residues within the enzyme and overall negative charge of the DNA. To decrease this affinity, the authors substituted key amino acids within the DNA-binding groove with neutral charged alanine. In doing so, they were able to design a new-and-improved Cas9, or enhanced SpCas9, that significantly decreased off-target binding without compromising on-target efficiency.
GenScript offers a variety of CRISPR constructs and services through a license with the Broad Institute:
- gRNA constructs: design gRNA sequences and clone into any vector, viral or non-viral, at just $199/clone.
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CRISPR gene editing improves the safety of porcine organs for human transplantation
November 16, 2015
Organ shortage for transplantation has been a critical problem for those that experience organ failure. Pig organs are ideal for organ replacement since they are the most comparable in size to humans; however, safety can be an issue for many patients. One reason for this incompatibility is the potential transmission of porcine endogenous retroviruses (PERV), which have the ability to infect human cells. So to address this problem and make pig organs safer for transplant, a group led by George Church at Harvard Medical School turned to the most effective gene editing technique available – CRISPR genome editing.
How were pig transplants made safer using CRISPR?
As a proof of principle, the authors examined the potential of knocking out all copies of the PERV polymerase gene in the porcine cell line PK15 using CRISPR/Cas9 technology. They found that the efficiency of the system was even better than expected: All 62 copies of the PERV pol gene were disrupted in the pig cells. In addition, when these edited cells were co-cultured with human cell lines, they found that there was a 1,000-fold reduction in PERV transmission. Together this report demonstrates the potential of CRISPR gene editing for eliminating harmful zoonoses from pig organs.
Genetically modified lab plants: the next source of powerful anti-cancer drugs?
October 19, 2015
Etoposide is a potent chemotherapeutic agent that has been cleared by the FDA in 1983 for the treatment of many types of cancers. Podophyllotoxin is a precursor for this drug, and has traditionally been derived from the Himalayan mayapple (Podophyllum hexandrum), a now endangered plant from the Indian Himalayan Region. To keep the future of this drug more certain, researchers have turned to genetic engineering and a common lab plant to become the new source of this important molecule.
What has become the newest supplier of this anticancer drug?
In this report, the authors first identified the biosynthetic genes that drive podophllotoxin production pathway. Ultimately, the team identified 10 enzymes that were important components of the pathway, and the genes for these enzymes were transiently expressed in the common lab plant Nicotiana benthiamiana via Agrobacterium. Expression of these enzymes drove the synthesis of (-)-4'-desmethylepipodophyllotoxin, another precursor to Etoposide. By enabling common plants to produce anticancer therapeutics, production of these important compounds has become more certain.
Yeast have been modified to synthesize opioids from sugar
September 28, 2015
Both natural opiates and semi-synthetic opioids have been classified by the World Health Organization as crucial for managing severe pain. Traditionally derived from the poppy plant, Papaver somniferum, opioids are used to produce morphine and thebaine, which are also precursors for other compounds such as codeine and hydrocodone. With changing climates and harvest yields, there is increased motivation to identify new sources of these chemical compounds. In a recent study published in Science, an unlikely source has been identified – yeast.
How were yeast engineered to synthesize opioids?
To synthesize opioids, yeast were engineered to generate precursor compounds from sugars. Genes from a variety of species, including poppy, rat, and bacteria, were integrated into the yeast genome. To make thebaine, 21 genes were knocked-in, and 23 genes were required to synthesize hydrocodone from sugar.
Does this mean yeast are the next target for producing illicit drugs?
Not quite. The methods described in this publication produce very low levels of hydrocodone – not enough to be clinically relevant, so improved scale-up methods would be needed for this to become commercial. Nonetheless, these discoveries highlight the important role microbes and gene editing play in the development of medicinal compounds.
T cell editing using CRISPR/Cas9 could revolutionize HIV therapeutics
September 15, 2015
Reinforcing the immune system by engineering lymphocytes to target and destroy viruses has the potential to be an effective therapy for many diseases. One potential approach to this strategy is to alter the genome of lymphocytes so that proteins that are typically hijacked by viruses are no longer present. While conceptually feasible, editing T cells has been challenging in practice; however, with the advent of mammalian cell editing using CRISPR/Cas9, T-cell editing is closer to becoming a reality.
How can CRISPR/Cas9 bring us closer to finding a cure for HIV?
In a study recently published in PNAS, scientists have optimized a protocol to introduce nucleotide replacements that would inhibit CXCR4 expression. The authors streamlined the CRISPR/Cas9 editing process by electroporating Cas9 ribonucleoproteins (RNPs) into CD4+ T cells. The RNPs, consisting of both a recombinant Cas9 enzyme and guide RNA, vastly improved editing efficiency, ultimately promoting knock-out of the CXCR4 cell-surface receptor. Taken together, these result suggest the potential of a new cell therapy approach for the fight against HIV.
Validating "predicted" regulatory elements through CRISPR editing of the non-coding genome
CRISPR/Cas9-mediated genome editing is not only an efficient way to create gene KO & KI, but is a uniquely powerful tool to functionally characterize the >98% of the genome that does not encode protein. A new study demonstrates how CRISPR can be used to systematically validate putative regulatory elements described by the ENCODE and EPIGENOME projects: even in a repeat-rich genomic region, a genomic insulator upstream of mouse tyrosinase was efficiently deleted or inverted, with no significant off-target effects and high efficiency in vivo, demonstrating a functional role for this noncoding region in regulating tyrosinase gene expression and mouse coat pigmentation.
How can CRISPR/Cas9 bring us closer to finding a cure for HIV?
GenScript makes it easy for your lab to start using CRISPR to create transgenic animals or isogenic cell lines. Our license with the Broad Institute and our extensive in-house experience making CRISPR-edited mammalian cell lines enable us to make your transition to CRISPR seamless:
- Brush up on CRISPR principles with webinars, protocols & FAQs
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Seruggia D et al. Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR–Cas9-mediated mutagenesis. Nucleic Acids Res. 2015 May 26;43(10):4855-67. Read the Free Full Text
CRISPR genome editing in human cells: improved targeting with the H1 promoter
A recent paper in Nature Communications reports success with a clever technique to make CRISPR-mediated genome editing easier in human cells. Compared to the commonly-used U6 promoter, driving guide RNA expression from the H1 promoter more than doubles the number of targetable sites within the genomes of humans and other eukaryotes.
Why is H1 more versatile than U6? The U6 promoter initiates transcription from a guanosine (G) nucleotide, while the H1 promoter can initiate transcription from A or G. In designing a gRNA sequence, the requirement for the protospacer adjacent motif (PAM) sequence "NGG" at the end of a 20-mer means that U6-driven gRNA must fit the pattern GN19NGG. But H1-driven gRNAs can also target sequences of the form AN19NGG, which occur 15% more frequently than GN19NGG within the human genome.
To support your genome editing efforts, GenScript offers:
||Ranganathan et al. Expansion of the CRISPR–Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs. Nat Commun. 2014 Aug 8;5:4516. Read Full Text
Making photosynthesis more efficient
Photosynthesis is such a fundamentally important process that you might think plants are highly efficient at it. However, scientists working to improve crop yields have noticed that one enzyme, Rubisco, represents a weak link in the photosynthetic pathway due to its poor oxygenase activity and slow turnover. A recent paper in Nature reports that plants can be engineered to use a better Rubisco enzyme from cyanobacteria, which increases carbon fixation rates. Tobacco plans whose native Rubisco gene was completely knocked out were able to grow by using transgenic cyanobacterial Rubisco, which successfully assembled into active enzyme to support autotrophic photosynthesis. The next step will be to introduce the remaining components of the cyanobacterial CO2 concentrating mechanism, including inorganic carbon transporters and carboxysome shell proteins, to recapitulate a complete and functional CCM in plants. If this strategy is successful in increasing total photosynthesis rates and thus improving crop yields, it could be a major breakthrough for sustainable farming and global food security.
Plant biologists are increasingly turning to CRISPR-mediated genome editing to create knock-in and knock-out strains, for several reasons. CRISPR is highly efficient with very low risk of off-target effects. It is easy to use in any lab, and compatible with widely-used methods for creating transgenic strains through agrobacterium-mediated transformation. However, CRISPR technology does not leave a ※transgenic§ footprint if the Cas9 and gRNA constructs are transiently expressed or are backcrossed out. Therefore there are hopes that new plant strains created using CRISPR/Cas9 genome editing technology may escape being labeled as GM strains which may increase their acceptance as food crops. Learn more about how gene synthesis and genome editing are being used in plant biology research applications.
GenScript is the global leader in gene synthesis to accelerate genetic engineering, CRISPR-mediated genome editing, recombinant enzyme expression, biosynthetic pathway engineering, and gene functional studies. We offer:
||Lin MT, Occhialini A, Andralojc PJ, Parry MA, Hanson MR. A faster Rubisco with potential to increase photosynthesis in crops. Nature. 2014 Sep 25;513(7519):547-50. Read Full Text
Will CRISPR bring us more nutritious fruit crops without GM worries?
Global nutrition and food security are major concerns as human population rises and the land area devoted to agriculture shrinks. Biotechnology has accelerated the development of improved food crops that can address these issues in order to boost the economic productivity of farms and improve human health. However, genetically modified food crops have met with consumer resistance and increasing regulation due in part to concerns over the long-term safety and environmental effects of transgenes being introduced into plants. One way in which the scientific community is responding is to identify new technologies that can achieve the nutrition and food security goal of prior genetic engineering efforts while avoiding the use of transgenes and the ※GM§ label that is controversial among consumers.
※Superbananas§ have made news headlines as a biofortified crop containing enhanced levels of vitamin A to counteract widespread vitamin A deficiencies in certain parts of the world. A new ※Science & Society§ paper in Trends in Biotechnology presents the argument that luxury items such as fruit crops may find more consumer acceptance for bioengineering than do staple crops such as grain and rice, and discusses the possibility that new technologies such as CRISPR-mediated genome editing may elude the regulatory designation as ※GMOs§ since they contain no foreign DNA.1 Successful CRISPR-mediated genome editing has been demonstrated in citrus fruits2 and a variety of other food crops and diverse plant species3.
GenScript offers gRNA constructs for CRISPR-mediated genome editing, including expert gRNA design for any species.
- Nagamangala K et al. Looking forward to genetically edited fruit crops. Trends Biotechnol. 2014 Aug 12. pii: S0167-7799(14)00147-4. Read Full Text.
- Jia H, Wang N. Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS One. 2014 Apr 7;9(4):e93806. Read Full Text.
- Jiang W. et al. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res. 2013 Nov 1;41(20):e188. Read Full Text.
Could CRISPR technology be used to cure AIDS and other devastating viral diseases?
Why are viral diseases like AIDS still incurable? Although antiretroviral drugs can effectively control viral load in many patients, the permanent integration of viral DNA into a host genome means that patients remain vulnerable to re-activation of a latent virus. Exciting new research now shows that CRISPR technology can remove HIV DNA that has integrated into the host genome in human cells, re-igniting our hopes for developing a true cure for AIDS.
CRISPR-mediated genome editing is revolutionizing biomedical research due to its precise targeting, high efficiency, and ease of use in any cell type or experimental system. CRISPR has been used to create new transgenic animal models for basic and translational research, and it holds promise for use in gene therapy and other medical applications.
GenScript's new GenCRISPR gRNA construct service makes it easy to perform CRISPR/Cas9-mediated genome editing in your own lab.
- Take advantage of complimentary gRNA design by our scientists who have demonstrated expertise in gRNA design: see our functionally-validated knock-out cell lines
- Our gene synthesis services have been cited in landmark publications in Nature Methods, Genetics, and Development by researchers who've pioneered CRISPR/Cas9 technology and applied it to new species: see references
CRISPR enables heritable multiplex genome editing in insects
Bmku70?knockout in B. mori creates a powerful new model for studying DNA repair
CRISPR/Cas9 technology for precise gene editing has already proven successful in mice, C. elegans, Xenopus tropicalis, and plants. Now CRISPR has been used the silkmoth Bombyx mori, an important insect model organism.
Ma?et al.?used CRISPR to disrupt the?Bmku70?gene, which is required for non-homologous end joining (NHEJ) and plays a role in telomere length maintenance, subtelomeric gene silencing and antigen diversity. Bmku70?knockouts exhibit an increased frequency of homologous recombination and thus can provide a powerful new model for future studies on the fundamental mechanisms of DNA repair.
Why is CRISPR more efficient than TALEN or ZFN for insect models?
Ma et al. report that numerous prior attempts at site-directed insertion of recombinant DNA into the?B. mori?genome have failed. These earlier experiments used ZFN or TALEN techniques, which have effectively generated transgenic strains of many species through targeted homologous recombination (HR). However, insect cells show a preference for the nonhomologous end joining (NHEJ) pathway rather than the HR pathway for repair of double strand breaks (DSBs), making the efficiency of HR-based genome editing very low.
While both zinc-finger nucleases (ZFN) and TAL effector nucleases (TALEN) can be engineering to target a locus of interest, they rely upon protein-DNA interaction; in contrast, CRISPR uses an RNA-DNA pairing to determine the specificity and activity of the nuclease. RNA-DNA interactions are generally much more stable than protein-DNA interactions, yielding higher efficiency of success. In addition, CRISPR is much simpler and faster for researchers to use.
The CRISPR method relies upon customized gene constructs that encode a codon-optimized Cas9 nuclease and a synthetic guide RNA for precise targeting. GenScript's gene synthesis service?can prepare the constructs you need for CRISPR/Cas9-based genome editing.