Nav: Home

Finding a rhyme and reason to CRISPR-Cas9's mutations

November 07, 2018

Since the early days of CRISPR-Cas9, researchers have known that this gene editing technology is excellent for breaking things. With precision, these scissor-like tools can be sent to any location in the genome to make a snip and break a gene. But exactly where and how CRISPR-Cas9 will disrupt the gene was anyone's guess. Until now. Investigators at Brigham and Women's Hospital, in collaboration with colleagues at the Broad Institute and MIT, have discovered that template-free Cas9 editing is predictable, and they have developed a machine learning model that can predict insertions and deletions with high accuracy. The team, led by co-corresponding authors Richard Sherwood, PhD, David Liu, PhD, and David Gifford, PhD, has demonstrated that this approach can be used to edit and repair mutations related to three diseases in human cell lines -- Hermansky-Pudlak syndrome, Menkes disease and familial hypercholesterolemia - with a predictable repair outcome in more than half of instances. These advancements have implications for both research and clinical applications. The team's findings are published this week in Nature.

"Like many projects, this one came out of a puzzling result: We wanted to use CRISPR-Cas9 to cause a random set of mutations at a particular spot in the genome, but we were finding that the mutations we were getting were far from random," said Sherwood, a principal investigator in the Brigham's Division of Genetics. "It turns out that the underlying sequence to which you are directing CRISPR-Cas9 allows you to predict, with a high degree of accuracy, which mutations you are most likely to get."

Many genetic diseases arise from insertions and deletions that disrupt a gene's function so being able to replicate these or fix them with accuracy would be a major coup. Conventional wisdom among CRISPR-Cas9 researchers has held that the tool randomly generates insertions and deletions in a gene unless researchers include a so-called repair template. But Sherwood's team has found that even without a template, one can predict which insertions and deletions are most likely to occur. At certain genomic sites, one particular mutation dominates - the team used the term "precise-50" to indicate when a single such mutation comprised more than 50 percent of all major editing products.

To conduct their project, Sherwood and colleagues, including Christopher Cassa, PhD, a principal investigator in the Brigham's Division of Genetics, constructed a library of 2,000 Cas9 guide RNAs (gRNAs) paired with DNA target sites. They used this library to train inDelphi, a machine learning model. They found that inDelphi could predict deletions of varying lengths and single base pair insertions with high accuracy (r = 0.87) in five human and mouse cell lines, and that it predicted up to 11 percent of the gRNAs were "precise-50."

To confirm these findings, the team used select gRNAs to correct mutations in cells collected from patients with genetic diseases that result from microduplications - a chromosomal change in which a small amount of genetic material gets duplicated. Hermansky-Pudlak syndrome, especially common in Puerto Ricans, causes blood clotting deficiency and albinism. Menkes disease results in copper deficiency. The team also generated cells with microduplications found in patients to result in familial hypercholesterolemia a disease in which LDL cholesterol levels are abnormally high. For all three diseases, delivering the appropriate Cas9 and guide RNA corrected the mutation with high efficiency.

The authors note that this work is still a proof-of-concept - while promising in cellular models in the lab, it requires further testing and additional steps to bring it into the clinic. In addition, only between 5 and 11 percent of Cas9 guide RNAs met the "precise-50" standard. Sherwood and the team will now work toward optimizing the efficiency of the guide RNAs by understanding why certain insertions or deletions are so much more common than others. They hope that others will leverage the tools they have developed to set regulatory standards for precision in existing therapeutic applications and expand what applications may be possible.

"Currently, most companies that are thinking about therapeutic applications for CRISPR-Cas9 are thinking about what genes you need to break to treat a disease. Our findings indicate that it's possible to predict where we may be able to repair mutations instead," said Sherwood. "We don't want to settle for 50 percent mutation correction - we want to continue to improve. Now that we know that CRISPR-Cas9 editing is predictable, we have a parameter to measure how to fix disease mutations even more precisely."
Funding for this work was provided by an NWO Rubicon Fellowship, NSF Graduate Research Fellowship, DARPA HR0011-17-2-0049, NIHRM1 HG009490L), R01 EB022376, R35 GM118062, HHMI, 1RO1HG008363, 1R01HG008754, 1K01DK101684), the Human Frontier Science Program, NWO, NSF, Brigham Research Institute, Harvard Stem Cell Institute, and American Cancer Society.

Brigham and Women's Hospital (BWH) is a 793-bed nonprofit teaching affiliate of Harvard Medical School and a founding member of Partners HealthCare. BWH has more than 4.2 million annual patient visits and nearly 46,000 inpatient stays, is the largest birthing center in Massachusetts and employs nearly 16,000 people. The Brigham's medical preeminence dates back to 1832, and today that rich history in clinical care is coupled with its national leadership in patient care, quality improvement and patient safety initiatives, and its dedication to research, innovation, community engagement and educating and training the next generation of health care professionals. Through investigation and discovery conducted at its Brigham Research Institute (BRI), BWH is an international leader in basic, clinical and translational research on human diseases, more than 3,000 researchers, including physician-investigators and renowned biomedical scientists and faculty supported by nearly $666 million in funding. For the last 25 years, BWH ranked second in research funding from the National Institutes of Health (NIH) among independent hospitals. BWH is also home to major landmark epidemiologic population studies, including the Nurses' and Physicians' Health Studies and the Women's Health Initiative as well as the TIMI Study Group, one of the premier cardiovascular clinical trials groups. For more information, resources and to follow us on social media, please visit BWH's online newsroom.

Brigham and Women's Hospital

Related Genetic Diseases Articles:

Screening for genetic diseases & chromosomal defects with a single biopsy improves pregnancy rates
Couples who are undergoing pre-implantation genetic diagnosis (PGD) in order to avoid transmission of inherited diseases, such as Duchenne muscular dystrophy or cystic fibrosis, should also have their embryos screened for abnormal numbers of chromosomes at the same time, according to research published in Human Reproduction journal.
New software tool could help doctors diagnose genetic diseases
An open-source software tool called Mendel,MD could help doctors analyze patients' genetic data in order to diagnose diseases caused by mutations.
Unveiling the bottlenecks to discovering the root causes of rare genetic diseases
A commentary paper including feedback from 40 scientists, says international cooperation is needed now more than ever; despite advances in technology and decades of research, the genetic mutations behind half of the 7,000 known rare genetic diseases in the world remain a mystery.
Unveiling how nucleosome repositioning occurs to shed light on genetic diseases
A research group led by a Waseda University professor became the first in the world to unveil the three-dimensional structure of an overlapping dinucleosome, a newly discovered chromatin structural unit.
Stem cell consortium tackles complex genetic diseases
Much of stem cell research over the past decade has focused on Mendelian disorders -- those caused by a single gene, such as cystic fibrosis, muscular dystrophy, and Huntington's disease.
Drosophila effectively models human genes responsible for genetic kidney diseases
The majority of genes associated with nephrotic syndrome (NS) in humans also play pivotal roles in Drosophila renal function, a conservation of function across species that validates transgenic flies as ideal pre-clinical models to improve understanding of human disease, a Children's National Health System research team reports in a recent issue of Human Molecular Genetics.
Stepping up the hunt for genetic diseases
The child's own genome thus consists of a maternal and a paternal genome.
Study of complex genetic region finds hidden role of NCF1 in multiple autoimmune diseases
Medical University of South Carolina investigators report pre-clinical research showing that a genetic variant encoded in neutrophil cystolic factor 1 (NCF1) is associated with increased risk for autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis, and Sjögren's syndrome, in the January 2017 issue of Nature Genetics.
Large Finnish genetic study uncovers potential new treatments for inflammatory diseases
Researchers from the Research Centre of Applied and Preventive Cardiovascular Medicine at the University of Turku, Finland, have studied over ten million DNA variations and found new links between the human genome and inflammation tracers.
Genetic risk factors for autism, MS and other diseases differ between the sexes
A pair of studies by researchers at UC San Francisco suggest that genetic variants that have distinct effects on physical traits such as height, weight, body mass, and body shape in men versus women are also linked to men's and women's risk for a range of diseases -- including autism, multiple sclerosis, type 1 diabetes, and others.

Related Genetic Diseases Reading:

Best Science Podcasts 2019

We have hand picked the best science podcasts for 2019. Sit back and enjoy new science podcasts updated daily from your favorite science news services and scientists.
Now Playing: TED Radio Hour

Failure can feel lonely and final. But can we learn from failure, even reframe it, to feel more like a temporary setback? This hour, TED speakers on changing a crushing defeat into a stepping stone. Guests include entrepreneur Leticia Gasca, psychology professor Alison Ledgerwood, astronomer Phil Plait, former professional athlete Charly Haversat, and UPS training manager Jon Bowers.
Now Playing: Science for the People

#524 The Human Network
What does a network of humans look like and how does it work? How does information spread? How do decisions and opinions spread? What gets distorted as it moves through the network and why? This week we dig into the ins and outs of human networks with Matthew Jackson, Professor of Economics at Stanford University and author of the book "The Human Network: How Your Social Position Determines Your Power, Beliefs, and Behaviours".