Transition to exhaustion: clues for cancer immunotherapy

Research on immune cells “exhausted” by chronic viral infection provides clues on how to refine cancer immunotherapy. The results were published Tuesday, Dec. 3 in Immunity. Scientists at Emory Vaccine Center, led by Rafi Ahmed, PhD, have learned about exhausted CD8 T cells, based on studying mice with chronic viral infections. In the presence of persistent virus or cancer, CD8 T cells lose much of their ability to fight disease, and display inhibitory checkpoint proteins Read more

Radiologists wrestle with robots - ethically

Emory bioethicist John Banja says: don’t believe the hype about AI replacing Read more

Opioids: crunching the Tweets

The aim is to be able to spot patterns of overdoses faster than prescription drug monitoring Read more

gene targeting

Manipulating mouse genes to order, CRISPR or old-school

Just a follow-up to last week’s announcement from the Emory Transgenic Mouse and Gene Targeting core that they are offering CRISPR/Cas9 gene editing for mice. Using CRISPR/Cas9 to produce genetically altered mice is a

Knockout_mice

Gene targeting – the 20th century way

substantial advance over the old way of doing knockouts and other manipulations (which itself won a Nobel Prize in 2007), mainly because it’s faster and easier.

To appreciate the difference, consider that the old way involves introducing DNA into mouse embryonic stem cells, and then selecting for the rare cells that take up and incorporate the DNA in the right way. Then the ES cells have to be injected into a blastocyst, followed by mouse breeding to “go germline.”

With CRISPR/Cas9, it’s possible to inject pieces of RNA that target the desired genetic changes, straight into a one-cell stage mouse embryo. Not every embryo has all the right changes, but the frequency is high enough to inject and screen. As this review explains, it’s possible to introduce mutations into three genes at once and get mice quickly, rather than make each one separately and then breed the mice together, which can take many months.

Also, because of the need for drug selection, the targeting construct in old-school gene targeting has to be a blunt instrument. That can make it hard to make subtle changes to a gene — like introduce point mutations corresponding to natural variations linked with human disease — without taking a sledgehammer to the entire gene locus. CRISPR/Cas9 takes care of that problem.

Despite the advantages of this technology, three things to keep in mind:

*Many genetically altered mice are already available “off the shelf” as part of the International Knockout Mouse/Mouse Phenotyping Consortium.

*Emory’s Mouse Core has been working with the company Ingenious Gene Targeting, and has been out-sourcing some of the tedious aspects of old-school gene targeting in mice to Ingenious, starting last year. Technicians there can generate a dazzling array of conditional knockouts. If you want your favorite gene to flip around and produce a fluorescent protein when you give the mice an antibiotic, but only in some cells — Ingenious can do that. Old school is actually still the way to go for fancy stuff like this.

*Jackson Labs in Maine also works with Emory, offering similar services, and offers a guarantee. Read more

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A CRISPR way to edit DNA

The CRISPR/Cas gene editing system has a lot of buzz behind it: an amusingly crunchy name, an intriguing origin, and potential uses both in research labs and even in the clinic. We heard that Emory scientists are testing it, so an explainer was in order.

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system was originally discovered by dairy industry researchers seeking to prevent phages, the viruses that infect bacteria, from ruining the cultures used to make cheese and yogurt. Bacteria incorporate small bits of DNA from phages into their CRISPR region and use that information to fight off the phages by chewing up their DNA.

At Emory, infectious disease specialist David Weiss has published research on CRISPR in some types of pathogenic bacteria, showing that they need parts of the CRISPR system to evade their hosts and stay infectious. Biologist Bruce Levin has modeled CRISPR-mediated immunity’s role in bacterial evolution.

What has attracted considerable attention recently is CRISPR/Cas-derived technology, which offers the ability to dive into the genome and make a very precise change. Scientists have figured out how to retool the CRISPR/Cas machinery – the enzymes that do the chewing of the phage DNA — into enzymes that can be targeted by an external guide.

For biologists in the laboratory, this is a way to probe a gene’s function by making an animal with its genes altered in a certain way. The method is gaining popularity here at Emory. Geneticist Peng Jin reports:

“CRISPR is much more efficient and quicker than traditional homologous recombination. One can directly inject the plasmid and guide RNA into mouse embryo to make knockout mice. You can also target multiple genes at the same time.”

The traditional method Jin refers to involves taking cultured embryonic stem cells, zapping DNA carrying a modified or disabled gene into them, and hoping that the cells’ repair machinery sews the DNA into the genome in the right way. Usually they have to use antibiotics and drugs to screen out all the cells where the DNA gets jammed into the genome haphazardly. Also, Jin adds that CRISPR/Cas technology can be used for whole-genome screens.

Tamara Caspary, a developmental biologist and scientific director of Emory’s transgenic mouse and gene targeting core, says she and her core team are in the process of developing and validating CRISPR, so that the technique could be accessible to many Emory investigators.

Potential clinical uses: Japanese scientists have proposed that CRISPR/Cas be employed against HIV infection. One can envision similar gene therapy applications.

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