Breaking News
August 17, 2018 - Give Your Child a Head Start With Math
August 17, 2018 - Ground-breaking study tests whether rejected livers can be made viable for transplantation
August 16, 2018 - New algorithm could improve diagnosis of rare diseases | News Center
August 16, 2018 - SCHILLER introduces latest generation of ECG device, CARDIOVIT AT-102 G2
August 16, 2018 - Proper treatment, refraining from smoking can reduce heart disease risk from type 2 diabetes
August 16, 2018 - Mount Sinai study could transform treatment for patients with retinal degenerative diseases
August 16, 2018 - Penn researchers develop first mouse model of idiopathic pulmonary fibrosis
August 16, 2018 - Four tips to help prevent fall allergy symptoms
August 16, 2018 - Women’s Preventive Services Initiative says screen all women annually for urinary incontinence
August 16, 2018 - At Stanford, patient discovers the source of her headaches, nausea | News Center
August 16, 2018 - To Prevent Injuries in Young Baseball Players, Chris Ahmad Reaches Out to Parents
August 16, 2018 - Restoring blood flow may be linked to longer survival in patients with critical limb ischemia
August 16, 2018 - New model of genetically engineered immune cells may help fight solid tumors
August 16, 2018 - Maternal stress increases anxious and depressive-like behaviors in female offspring
August 16, 2018 - Childhood exposure to secondhand smoke increases risk of COPD death in adulthood
August 16, 2018 - Scientists uncover key control mechanism of DNA replication
August 16, 2018 - NIH begins first-in-human trial of experimental live, attenuated Zika virus vaccine
August 16, 2018 - Two diabetes medications don’t slow progression of type 2 diabetes in youth
August 16, 2018 - 5 Questions: How Stanford research is making MRI scans safer for kids | News Center
August 16, 2018 - Columbia Celebrates 25th Anniversary of White Coat Ceremony
August 16, 2018 - Phonak’s new smallest and most discreet Virto B-Titanium hearing aid
August 16, 2018 - New project aims to study growth of water-based microorganisms
August 16, 2018 - Higher social dominance linked to faster decision-making in men
August 16, 2018 - Blood test in early pregnancy could determine a woman’s later risk for gestational diabetes
August 16, 2018 - New research confirms link between DDT exposure and autism
August 16, 2018 - Neurodevelopmental Anomalies, Birth Defects Linked to Zika ID’d
August 16, 2018 - Risk of heart failure up in ALVSD patients with diabetes
August 16, 2018 - Exercise reduces symptoms and fatigue in patients with chronic kidney disease
August 16, 2018 - Study reveals role of RUNX proteins in DNA repair
August 16, 2018 - New research finds no harm from average salt consumption
August 16, 2018 - Researchers develop new way of testing bacterial resistance to antibiotics
August 16, 2018 - Magnetic gene in aquarium fish could open doors to treatment for epilepsy, Parkinson’s
August 16, 2018 - Five tips for successful long-term breastfeeding
August 16, 2018 - Researchers identify brain networks involved in object naming
August 16, 2018 - Promoting HPV Vaccine Doesn’t Prompt Risky Sex by Teens: Study
August 16, 2018 - Treating Rheumatoid Arthritis: Search for a Cure
August 16, 2018 - Research shows in the long run, charcoal toothpaste likely won’t whiten teeth
August 16, 2018 - Seattle Children’s opens new clinic to provide convenient access to pediatric specialty care services
August 16, 2018 - Curious case of the lost contact lens
August 16, 2018 - GN Hearing unveils world’s first Premium-Plus hearing aid
August 16, 2018 - Parental life span linked with increased longevity and health in daughters
August 16, 2018 - Health leaders reveal ten most important medicines in NHS history
August 16, 2018 - Mobile health devices diagnose hidden heart condition in at-risk populations
August 16, 2018 - When it comes to shedding pounds, it pays to think big
August 16, 2018 - Liva Healthcare announces appointment of Thomas Cooke as clinical services manager in the UK
August 16, 2018 - New digital pharmacy aims to help people living with chronic care conditions
August 16, 2018 - Preventing ACL injuries in high school athletes
August 16, 2018 - Experts provide insight into novel concepts and approaches for stroke rehabilitation
August 16, 2018 - Scientists reverse congenital blindness in mouse model
August 16, 2018 - Study shows link between use of benzodiazepines and increased risk of Alzheimer’s disease
August 16, 2018 - Study provides new insight into how ‘trash bag of the cell’ traps and seals off waste
August 16, 2018 - Trial shows PARP inhibitor as novel treatment option for patients with advanced breast cancers
August 16, 2018 - Prenatal exposure to violence increases toddlers’ aggressive behavior to their mothers
August 16, 2018 - Can manipulating gut microbes improve cardiac function in patients with heart failure?
August 16, 2018 - Hearts of newborn piglets can completely heal after heart attacks
August 16, 2018 - Ablating the mutant p53 gene in mice with colorectal cancer inhibits tumor growth
August 16, 2018 - Higher BMI in people with prediabetes related to evening preference and lack of sufficient sleep
August 16, 2018 - Using peripheral nerve blocks to treat facial pain may produce long-term pain relief
August 16, 2018 - Neural stem cells are the key to tail regeneration
August 16, 2018 - Study compares genetic and neural contributions to ADHD in children with or without TBI
August 16, 2018 - Adding energy drinks to alcohol may exacerbate negative effects of binge drinking
August 16, 2018 - Eye Examination Can Help Detect Abuse in Children
August 16, 2018 - Know the Difference: Rheumatoid Arthritis or Osteoarthritis?
August 16, 2018 - From ‘sea of mutations,’ two possible cancer links rise to the surface
August 16, 2018 - Does medical school take too long?
August 16, 2018 - Brown University researchers reveal key physical properties of ‘giant’ cancer cells
August 16, 2018 - Regular resistance training improves exercise motivation
August 16, 2018 - Feds urge states to encourage cheaper plans off the exchanges
August 16, 2018 - Seven activities that prevent you from getting quality sleep during summer
August 16, 2018 - Five ways to tell if your baby is getting enough milk from breastfeeding
August 16, 2018 - From Pigs to Peacocks, What’s Up With Those ‘Emotional-Support Animals’?
August 16, 2018 - Breast cancers enlist the help of normal cells to help them spread and survive
August 16, 2018 - Engaging with “high-need” patients outside the clinic
August 16, 2018 - Research illuminates how online forum may offer suicide prevention support for males
August 16, 2018 - Researchers identify way to grow immune cells at large scale for preventing cancer reoccurrence
August 15, 2018 - Keck Medicine of USC’s hospitals ranked among nation’s best for the 10th consecutive year
August 15, 2018 - Researchers compare existing approaches for automating diagnostic procedures of skin lesions
August 15, 2018 - Autism risk determined by health of mom’s gut, research reveals
August 15, 2018 - WELL for Life challenges you to explore the great outdoors
August 15, 2018 - ‘Zombie’ gene protects elephants from cancer, study finds
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