Breaking News
June 19, 2018 - After addiction, the long road back to good health
June 19, 2018 - High blood pressure could be an early sign of dementia
June 19, 2018 - Innovative drugs and new European treatment guidelines refine, improve MS therapy
June 19, 2018 - BIDMC scientists develop new tool to benefit patients with HCV-associated liver failure
June 19, 2018 - Diabetes diagnosis may come with increased risk of pancreatic cancer for African-Americans, Latinos
June 19, 2018 - Personalized Goals, Cash Motivate Heart Patients to Exercise
June 19, 2018 - Nipah Virus (NiV) | CDC
June 19, 2018 - Genomics offers new treatment options for infants with range of soft tissue tumors
June 18, 2018 - Study shows how moderate consumption of alcohol can protect the heart
June 18, 2018 - Gene editing technology predicts heart disease risk
June 18, 2018 - Who Will and Who Won’t Get the Flu?
June 18, 2018 - Research shows effective responses to online feedback
June 18, 2018 - Scientists to focus on big data and genetics to identify risk factors for dementia
June 18, 2018 - Ultrasound-based technology for assessing overweight adolescents with liver disease
June 18, 2018 - Osteochondral knee defect treated using cell technology
June 18, 2018 - Study demonstrates increased levels of gum disease in people at risk of rheumatoid arthritis
June 18, 2018 - Ebola & Marburg | NIH: National Institute of Allergy and Infectious Diseases
June 18, 2018 - Brains, eyes, testes: off-limits for transplants?
June 18, 2018 - Drug used to treat myelofibrosis can awaken ‘dormant’ lymphomas in the bone marrow
June 18, 2018 - New study focuses on best, cost effective practices to bridge treatment gap for brain disorders
June 18, 2018 - New study highlights predictors that prevent from achieving remission in early RA
June 18, 2018 - Neuroscientists map feeling of cool touch to the brain’s insula in mouse model
June 18, 2018 - Study highlights potential use of blood biomarkers as diagnostic tool for sleep apnea
June 18, 2018 - Eating plant-based diet can reduce risk for heart problems in people with type 2 diabetes
June 18, 2018 - Lenabasum has acceptable safety and tolerability in diffuse cutaneous systemic sclerosis
June 18, 2018 - Study shows link between risky opioid prescriptions and increased odds of death
June 18, 2018 - Bone density scans could help determine likelihood of cardiovascular disease
June 18, 2018 - Mechanical thrombectomy appears to be important therapy for acute stroke in very old patients
June 18, 2018 - Novel compound as effective as FDA-approved antibiotics for treating deadly infections
June 18, 2018 - Ironwood Pharmaceuticals Announces FDA Orphan Drug Designation for Olinciguat for the Treatment of Sickle Cell Disease
June 18, 2018 - Surgical outcomes equivalent whether physician anesthesiologist assisted by nurse anesthetist or AA
June 18, 2018 - Studies provide insight into molecular changes prior to onset of arthritis
June 18, 2018 - Dyaco unveils specialist medical and rehabilitation equipment range in the UK
June 18, 2018 - Engineers develop algorithm to monitor joints of patients with arthritis
June 18, 2018 - Women with higher vitamin D blood levels have lower risk for breast cancer
June 18, 2018 - New studies help elucidate role of sleep in chronic pain
June 18, 2018 - Researchers link red meat sensitivity spread by ticks with heart disease
June 18, 2018 - Research explores role of autopsy in cardiovascular medicine
June 18, 2018 - Motif Bio Submits NDA for Iclaprim
June 18, 2018 - NIH-funded researchers identify target for chikungunya treatment
June 18, 2018 - Negative emotions are murkier, less distinct in adolescence
June 18, 2018 - Gut microbiome may be potential contributor to depression, anxiety in people with obesity
June 18, 2018 - Canakinumab reduces gout rate by more than half in atherosclerosis patients, study shows
June 18, 2018 - What Does the Future Hold?
June 18, 2018 - FDA Approves Keytruda (pembrolizumab) for Treatment of Refractory or Relapsed Primary Mediastinal Large B-Cell Lymphoma (PMBCL)
June 18, 2018 - School cliques don’t always click
June 18, 2018 - Three experts from The Tinnitus Clinic contribute to major review on pulsatile tinnitus
June 18, 2018 - Unwieldy health costs often stand between teachers and fatter paychecks
June 18, 2018 - Link between frailty and mortality remains unchanged despite lower death rates, study finds
June 18, 2018 - Sleep disorders appear to be first sign of serious neurological diseases
June 18, 2018 - Childhood, adult obesity raise risk of developing hip and knee osteoarthritis
June 18, 2018 - Study unravels ‘blood stem cell niche’ puzzle
June 18, 2018 - People with heart problems do not take enough exercise, shows study
June 18, 2018 - Strong Link Identified Between T2DM and Parkinson’s Disease
June 18, 2018 - Early childhood interventions show mixed results on child development
June 18, 2018 - Chronic use of opioids related to increased risk of fracture nonunion
June 18, 2018 - AHA: Family Perseveres After Losing Father to Heart Attack
June 18, 2018 - Oral propranolol seems safe for infantile hemangioma
June 18, 2018 - New study gives explanation for food’s prominence in memory
June 18, 2018 - First photoactive drug to fight Parkinson’s disease
June 18, 2018 - Patient’s self-evaluation of personality disorders not as far off as previously perceived
June 18, 2018 - Upadacitinib Monotherapy Meets All Primary and Ranked Secondary Endpoints Versus Methotrexate in a Phase 3 Study in Rheumatoid Arthritis
June 18, 2018 - Vitamin B3 has a positive effect on damaged nerve cells in Parkinson’s patients
June 17, 2018 - Sunovion Announces FDA Acceptance of New Drug Application for Apomorphine Sublingual Film (APL-130277)
June 17, 2018 - Bid to beat obesity focuses on fat that keeps us warm
June 17, 2018 - Work Stress May Increase Risk of Developing Atrial Fibrillation
June 17, 2018 - New Zealand’s secret recipe for active school travel: The neighborhood built environment
June 17, 2018 - New Medscape report reveals sexual harassment rate of physicians
June 17, 2018 - Surgical Blood Transfusions Tied to Clot Risk
June 17, 2018 - CDC chief makes $375K, far exceeding his predecessors’ pay
June 17, 2018 - Education linked to higher risk of short-sightedness
June 17, 2018 - Patients with Moderate-to-Severe Ulcerative Colitis Achieved Clinical and Endoscopic Remission with Mirikizumab in Phase 2 Trial
June 17, 2018 - UA registers a more customised multifocal lens to correct presbyopia
June 17, 2018 - U.S. FDA and European Medicines Agency Accept Regulatory Submissions for Review of Talazoparib for Metastatic Breast Cancer Patients with an Inherited BRCA Mutation
June 17, 2018 - Vaginal estrogen tablets, moisturizers and placebo gel all can improve vaginal discomfort
June 17, 2018 - Addition of Bezafibrate Beneficial in Primary Biliary Cholangitis
June 17, 2018 - Radiation Therapy for Cancer – National Cancer Institute
June 17, 2018 - Technology could help pregnant women detect health complications
June 17, 2018 - Study finds drop in frequent use of ED after Affordable Care Act
June 17, 2018 - Do Antipsychotic Meds for Kids Raise Diabetes Risk?
CRISPR-Cas9-based strategy allows researchers to precisely alter hundreds of different genes

CRISPR-Cas9-based strategy allows researchers to precisely alter hundreds of different genes

image_pdfDownload PDFimage_print

Geneticists have been using model organisms ranging from the house mouse to the single-cell bakers’ yeast, Saccharomyces cerevisiae, to study basic biological processes that regulate human development and physiology, and that can be compromised in various diseases. This has been possible because many of the genes that control these processes in humans are also present with similar functions in those other species; and because genes in model organisms can be mutated and deleted in the laboratory at will. Thus far, however, even in easy-to-manipulate yeast, genes had to be deleted one-gene at a time, often with additional undesired sequence modifications left behind in their genome.

A team at Harvard’s Wyss Institute led by its Core Faculty member George Church now presents a CRISPR-Cas9-based strategy in Nature Biotechnology that solves both of these problems. Using baker’s yeast, the researchers developed a high-throughput approach that allows researchers to precisely alter hundreds of different genes or features of a single gene at once in individual yeast cells with 80 to 100% efficiency, select cells from the population that show specific behaviors, and identify the gene alterations that either trigger or prevent them.

“Our method not only offers a more efficient and precise way to perform high-throughput “functional genomics” in yeast than what was possible with previous methods. It will also allow us to model and test subtle human gene variations in yeast cells that have been loosely associated with certain traits or disorders, and find out which ones may actually be relevant,” said Church, Ph.D., who also is Professor of Genetics at Harvard Medical School (HMS) and of Health Sciences and Technology at Harvard and the Massachusetts Institute of Technology (MIT).

Variations in human genes normally do not occur as perfect deletions of their sequences from the genome, but rather consist of small point mutations — substitutions of single A, T, C, or G base units in the DNA code for one of the other ones — or the insertion or deletion of a few base units. To recreate such variations in the yeast genome in the absence of other potentially interfering variations, the team leveraged CRISPR-Cas9, which can be precisely targeted to pre-selected sequences in the DNA with the help of a small guide RNA (sgRNA). After the Cas9 enzyme has cut its target sequence, a process known as homology-directed recombination (HDR) can repair the gene by using information from an additionally supplied donor template sequence that carries a variation of interest.

“We have developed a strategy that physically links the blueprints for sgRNA and donor template in one stable and heritable extra-chromosomal DNA molecule (guide+donor). This enabled us to construct large libraries of variants in one reaction, deliver multiple corresponding sgRNAs and donor templates en masse to yeast cells, and identify those that stimulate a certain cell behavior by next-generation sequencing,” said Postdoctoral Fellow Xiaoge Guo, Ph.D., one of the study’s first authors.

In proof-of-concept studies, the team first focused on a single highly conserved gene encoding the DNA helicase and repair enzyme SGS1. They then broadly damaged the DNA of the yeast cell population carrying the guide+donor library with a toxic reagent, and sequenced the DNA of the surviving cells. This allowed them to uncover mutations compromising SGS1 features that are vital for the repair of damaged DNA and that guarantee the cells’ continued survival.

Next, the team applied their guide+donor strategy to delete 315 members of a poorly understood gene family, which encode so-called small open reading frames (smORFs) that are scattered throughout the genome, in one fell swoop. By analyzing how this affects yeast cells’ survival in different environmental stress conditions, they could assign previously unknown, essential functions to specific smORFs, thereby opening a new gateway into their analysis.

“Besides using the method to tease new functions out of genes and larger gene families, an intriguing potential also lies in the investigation of non-coding sequences in the genome to advance our understanding of gene regulation and chromosome biology,” said first and co-corresponding author Alejandro Chavez, M.D., Ph.D., who as a Postdoctoral Fellow was co-mentored by Church and Wyss Institute Core Faculty member James Collins and is now Assistant Professor at Columbia University.

Collins, Ph.D., who collaborated with the team on the study, is also the Termeer Professor of Medical Engineering & Science at MIT and a Professor of Biological Engineering at MIT. “We can also use the guide+donor method for synthetic biology applications that aim to engineer yeast cells with specific metabolic and industrially relevant abilities, or transfer it to pathological yeast strains for the discovery of genes and gene functions that affect their infectious properties,” he said.

“This newest application of the CRISPR-Cas9 technology that emerged through a dynamic collaboration between the Church and Collins labs opens yet another path towards discovery of previously hidden molecular mechanisms by which cells regulate their physiology, and when dysregulated, lead to infections as well as human disease,” said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at HMS and the Vascular Biology Program at Boston Children’s Hospital, as well as Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

Source:

https://wyss.harvard.edu/profiling-the-genome-hundreds-of-variations-at-a-time/

Tagged with:

About author

Related Articles