Nav: Home

Better genome editing

August 21, 2018

A major obstacle to in-cell genome editing is, well, the cell itself.

"Human cells don't like to take in stuff," explained UC Santa Barbara's Norbert Reich, a professor in the Department of Chemistry and Biochemistry. The human cell has evolved a "trash disposal" mechanism that isolates and breaks down foreign proteins and other unwanted biomolecules, pathogens and even damaged cellular structures, he explained. So, for people in fields such as biotechnology, biopharmacology and genomic research and therapeutics -- such as those working with the gene editing juggernaut CRISPR-Cas9 technology -- results are only as good as their ability to efficiently bypass this defense mechanism and accurately introduce proteins into animal cells.

Reich and his team have developed such a method. Their technique, estimated to be 100 to 1,000 times more efficient than current methods, gives users complete spatiotemporal control of the genome editing delivery, in effect allowing them to decide exactly when and where to release genome editing proteins.

"We can actually hit individual cells," Reich said. "We can even hit parts of a cell so we could release the protein into only a part of the cell. But the main point is that we have the control over where and when this protein that's going to cut the DNA is going to be released."

The research by Reich's group, "Light-Triggered Genome Editing: Cre Recombinase Mediated Gene Editing with Near-Infrared Light" appears in the journal Small.

One attention-grabbing recent breakthrough in biotechnology is the use of gene editing proteins -- "molecular scissors" such as CRISPR, Cas and, in this study, Cre -- to find, cut and paste specific sections of target DNA sequences. Originally a defense mechanism used by bacteria and archaea to recognize DNA from attacking viruses and mark them for destruction, scientists have since developed methods of recognizing, cutting and binding base pair sequences of various lengths, using various proteins. The potential for this technology is massive, and ranges from basic research that determines the function and identification of genes to therapies that could fix cellular-level defects.

Key to the Reich group's light-triggered genome editing are hollow gold nanospheres onto which are coated DNA reporter strands (they fluoresce red) and a protein fusion of Cre recombinase and cell-penetrating peptides. And near-infrared light.

"So now we've got a homing device and a delivery agent," Reich said, explaining that the Cre recombinase and peptide fusion act as the targeting system, one that goes into play when the target cell does its cellular trash disposal.

Once taken into the cell, the nanoshell is enveloped in an endosome -- a membranous pocket that isolates it and transports it through the cell.

"But the nanoshells don't do anything because they're entrapped," Reich said. Ultrafast pulsed near-infrared laser light -- which is harmless to cells and is efficient at tissue penetration -- is then aimed at the entrapped nanoshells and their protein coats.

"Near-infrared wavelengths cause a really interesting thing to happen," Reich said. "It causes the gold nanoshell to get excited and it causes whatever we've attached to come off." At the same time, nanobubbles form, causing openings in the endosome and allowing its protein contents to escape. The proteins are now free to home in on the cell's nucleus, where its genetic material is stored, and gain entry with the cell-penetrating peptide. And Cre can get to work finding, cutting and pasting its reporter strands into the helix.

The group's in-vitro experiment proved successful: After a period of incubation, cells penetrated by the protein-coated nanoshells, followed by irradiation, glowed red.

"We didn't engineer anything that would make the cells behave differently," Reich said. "We made it so the cell would look different because of this fluorescent protein."

Said the paper's lead author, Dean Morales, who is now a postdoctoral researcher at Los Alamos National Laboratory, "As a basic research tool, with spatiotemporal control each cell can become an experiment. Imagine you'd like to study the function of a certain gene and how it alters that cell's behavior or its behavior with a close neighbor. Using the plasmonic nanoparticles as an antenna we can either turn on or turn off a gene of interest and observe in real-time the ramifications of its activity."

Spatiotemporal control also allows those who employ it to tread lightly on DNA, the rewriting of which, the researchers acknowledge, has very powerful and transgenerational effects.

"In certain cases, like somatic mutations, not every cell in the body would require editing," Morales said. "The ability to control where and when the editing machinery can be used provides transience to the procedure. The importance of this is that current approaches to gene editing often result in the editing machinery being left in an active form in the targeted cell, with unknown long-term ramifications. Our approach delivers the editing machinery in a transient fashion, and thus circumvents this problem."
-end-
Research on this project was conducted also by co-authors Erin Morgan, Megan McAdams and Amanda B. Chron of UC Santa Barbara; and Jeong Eun Shin and Joseph Zasadzinski, of University of Minnesota.

University of California - Santa Barbara

Related Dna Articles:

A new twist on DNA origami
A team* of scientists from ASU and Shanghai Jiao Tong University (SJTU) led by Hao Yan, ASU's Milton Glick Professor in the School of Molecular Sciences, and director of the ASU Biodesign Institute's Center for Molecular Design and Biomimetics, has just announced the creation of a new type of meta-DNA structures that will open up the fields of optoelectronics (including information storage and encryption) as well as synthetic biology.
Solving a DNA mystery
''A watched pot never boils,'' as the saying goes, but that was not the case for UC Santa Barbara researchers watching a ''pot'' of liquids formed from DNA.
Junk DNA might be really, really useful for biocomputing
When you don't understand how things work, it's not unusual to think of them as just plain old junk.
Designing DNA from scratch: Engineering the functions of micrometer-sized DNA droplets
Scientists at Tokyo Institute of Technology (Tokyo Tech) have constructed ''DNA droplets'' comprising designed DNA nanostructures.
Does DNA in the water tell us how many fish are there?
Researchers have developed a new non-invasive method to count individual fish by measuring the concentration of environmental DNA in the water, which could be applied for quantitative monitoring of aquatic ecosystems.
Zigzag DNA
How the cell organizes DNA into tightly packed chromosomes. Nature publication by Delft University of Technology and EMBL Heidelberg.
Scientists now know what DNA's chaperone looks like
Researchers have discovered the structure of the FACT protein -- a mysterious protein central to the functioning of DNA.
DNA is like everything else: it's not what you have, but how you use it
A new paradigm for reading out genetic information in DNA is described by Dr.
A new spin on DNA
For decades, researchers have chased ways to study biological machines.
From face to DNA: New method aims to improve match between DNA sample and face database
Predicting what someone's face looks like based on a DNA sample remains a hard nut to crack for science.
More DNA News and DNA Current Events

Trending Science News

Current Coronavirus (COVID-19) News

Top Science Podcasts

We have hand picked the top science podcasts of 2020.
Now Playing: TED Radio Hour

Sound And Silence
Sound surrounds us, from cacophony even to silence. But depending on how we hear, the world can be a different auditory experience for each of us. This hour, TED speakers explore the science of sound. Guests on the show include NPR All Things Considered host Mary Louise Kelly, neuroscientist Jim Hudspeth, writer Rebecca Knill, and sound designer Dallas Taylor.
Now Playing: Science for the People

#576 Science Communication in Creative Places
When you think of science communication, you might think of TED talks or museum talks or video talks, or... people giving lectures. It's a lot of people talking. But there's more to sci comm than that. This week host Bethany Brookshire talks to three people who have looked at science communication in places you might not expect it. We'll speak with Mauna Dasari, a graduate student at Notre Dame, about making mammals into a March Madness match. We'll talk with Sarah Garner, director of the Pathologists Assistant Program at Tulane University School of Medicine, who takes pathology instruction out of...
Now Playing: Radiolab

Kittens Kick The Giggly Blue Robot All Summer
With the recent passing of Ruth Bader Ginsburg, there's been a lot of debate about how much power the Supreme Court should really have. We think of the Supreme Court justices as all-powerful beings, issuing momentous rulings from on high. But they haven't always been so, you know, supreme. On this episode, we go all the way back to the case that, in a lot of ways, started it all.  Support Radiolab by becoming a member today at Radiolab.org/donate.