Snip Vs. Shred: CRISPR, CAS9, CAS3 and Cascade

Snip Vs. Shred: CRISPR, CAS9, CAS3 and Cascade

By Alexander Gelfand, for the Johns Hopkins School of Public Health Magazine.
© Johns Hopkins University

The powerful genome-editing technology known as CRISPR-Cas made headlines this year—partly because many leading biologists called for a moratorium last March against using it to modify the genomes of human embryos, only to discover in April that Chinese scientists had already done just that.

But CRISPR-Cas is more than a genetic engineering tool with profound ethical implications. In fact, the tool itself is a modified version of one of several types of naturally occurring bacterial immune systems that fight the viruses known as bacteriophages. The particular system that researchers have adapted for gene-editing is a relatively rare and simple one called CRISPR-Cas9. Scott Bailey, PhD, Associate Professor in the Department of Biochemistry and Molecular Biology, recently described the atomic structure of a far more common, and far more complicated, CRISPR-Cas system called CRISPR-Cascade—one with profound implications of its own.

Bacteria use CRISPR-Cas systems to store the genetic signatures of phages that have previously infected them. These viral mugshots appear as stretches of DNA separated by short, repeated sequences. Together, the viral DNA and the repeats form arrays called clustered, regularly interspaced, short palindromic repeats, or CRISPRs. When a phage invades, CRISPR-Cas uses short strings of RNA to compare the phage’s DNA with its archive of viral signatures. If it finds a match, specialized CRISPR-associated (Cas) proteins are dispatched to disable the invader through various means. Those means depend on the particular flavor of CRISPR-Cas system involved: The Cas9 protein cuts neatly through phage DNA like a pair of molecular scissors; Cas3, employed by CRISPR-Cascade, chews it up like a shredder.

Using synthetic RNA, scientists can program CRISPR-Cas9 to target specific genes, allowing them to disrupt, delete, or replace DNA more quickly, easily, and cheaply than ever before. And whereas making changes to multiple genes at the same time was once extremely difficult and inefficient, CRISPR-Cas9 makes it simple. Its reliability and ease of use have already revolutionized genomic research, and could one day lead to clinical applications such as gene therapy.

Bailey, meanwhile, says that understanding how CRISPR-Cascade fends off phages could help scientists design microbes that are super-resistant to viruses. That would be a boon the pharmaceutical industry, which uses genetically engineered bacteria and yeast to produce a variety of drugs, including insulin. Learning Cascade’s secrets could also help scientists weaken the bacterial immune system in order to kill harmful microbes, better understand (and therefore prevent) antibiotic resistance—and ultimately generate new ways of grappling with bacteria that researchers have not yet even begun to imagine.

The CRISPR-Cas system was discovered by dairy-industry researchers who wanted to stop phages from ruining the bacterial cultures that are used to make cheese and yogurt.

CRISPR-Cas9 has worked everywhere it has been applied—from wheat and trees to monkeys and mice. In laboratory experiments on human cells, researchers have used it to remove HIV DNA from a human genome and to fix a mutation that causes cystic fibrosis.

Cascade stands for “CRISPR-associated complex for antiviral defense.” Bailey and doctoral student Sabin Mulepati visualized CRISPR-Cascade’s large, complex structure using a technique called x-ray crystallography—and a particle accelerator housed at Stanford University.

Diacylglycerol and PIP3 Signaling

Diacylglycerol and PIP3 Signaling

Creating cover art and illustrations for MCP, by Rajendrani Mukhopadhyay: ASBMB Today, January 2014

The way Fairman worked on the art for the MCP special issue on posttranslation modications was typical for any project she does. She met with Gerald Hart of Johns Hopkins University, the MCP associate editor overseeing the issue, and ASBMB’s publications director, Nancy Rodnan, whose idea it was to hire a professional medical illustrator. Hart explained the science in the various articles. With input from Mary Chang, MCP’s managing editor, the group focused on the images that were either schematics or illustrations. They left alone the images that were captured by a camera or a computer.

“One of the things that I strived to do for this journal was to come up with a consistent style,” explains Fairman. For elements that came up repeatedly, such as ubiquitination, acetylation, proteins and organelles, Fairman established a style so that all of the figures throughout the special issue had the same look and feel. Fairman also says she stuck to scientific conventions as much as possible in terms of colors and symbols. “For example, thinking back to my time in organic chemistry in undergrad, in the little molecular model set, oxygen is usually red, carbon is black, and hydrogen is white,” she says. “Whenever we create any visual, we have to keep in mind who the audience is. Because MCP has a scientific audience, I’ve tried to come up with conventions that people are used to seeing.”

Fairman says it can be a challenge to figure out what should be kept in and left out of an illustration. She had a difficult case with one of the figures from the MCP special issue. “e illustration shows a really complicated mechanism, where these different proteins on the cell membrane, endoplasmic reticulum, nucleus, all the different organelles, are interacting with each other,” she says. “Instead of showing every single protein in its correct configuration, the best thing to do to drive home the message is to use color coding. Not worry so much about what those proteins actually look like but focus more on what they do.”

With the cover, Fairman took another tack, because the cover has a different role than figures in the scienfitic articles. The inspiration for the cover art came from figure 1 in the article by Corina Antal and Alexandra C. Newton at the University of California, San Diego, on the dynamics of lipid second messenger phosphorylation. “e cover isn’t necessarily meant to show the whole mechanism in a way that the readers will completely understand it,” says Fairman. “It is supposed to engage them and bring them into the journal, wanting to read that featured article.”

The Philadelphia Chromosome

The Philadelphia Chromosome

This illustration was created for JAAPA, February 2013 • 26(2).

The Philadelphia chromosome is formed when a piece of chromosome 9 exchanges places with a piece of chromosome 22, resulting in a balanced translocation t(9;22)(q34;q11) and the formation of an abnormal fusion gene BCR-ABL1.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm. The American Cancer Society estimated that in 2012, the number of newly diagnosed CML cases would be 5,430, with 610 deaths.1 The discovery of the tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment and prognosis of CML, making imperative early recognition of CML. Timely diagnosis will ensure that patients receive early treatment, significantly improving their prognosis.

CML is caused by a cytogenetic abnormality that is thought to be acquired and involves a balanced translocation between chromosome 9 and chromosome 22. The BCR gene on chromosome 22 fuses with the ABL1 gene on chromosome 9, resulting in the abnormal fusion gene BCR-ABL1 located on chromosome 22 (Figure 1). The fusion gene produces a protein with tyrosine kinase activity that causes cells to proliferate. The resultant abnormal chromosome 22 [t(9;22)(q34;q11)] is also known as the Philadelphia (Ph) chromosome. It is identified in 95% of patients with CML and can be detected in all myeloid cells, including granulocytes, erythrocytes, monocytes, and B lymphocytes

SomaLogic Somamer MOA

SomaLogic Somamer MOA

This explanatory animation illustrates SomaLogic’s SOMAmer technology. At the heart of SomaLogic’s unique platform are SOMAmer (Slow Off-rate Modified Aptamer) modified nucleic acid-based protein binding reagents, each of which are highly specific for their cognate protein. To date, SomaLogic has developed SOMAmers to a broad array of over 1000 different protein targets critical to normal and disease biology, and continue to add new SOMAmers all the time.

SOMAscan™ technology (illustrated below) takes advantage of two distinctive properties of SOMAmers: Their specific protein-binding properties and their primary nucleic acid sequences. These two properties not only ensure accurate protein detection and measurement, but allow the multiplexing of literally up to more than a thousand such measurements in each of several hundred different samples in a single experiment.

somascan

This technology ensures that measurements are taken only of proteins specific to the SOMAmers being used, giving an accurate readout of both protein identity and concentration in the original sample. The ability to detect anywhere from a few to more than a thousand proteins in literally hundreds of different samples a day – depending on the requirements of the analysis being undertaken – provides for a quick determination of a protein biomarker “signature” indicative of the disease or biological state (e.g., drug response) being studied.

SomaLogic Somamer MOA from Fairman Studios on Vimeo.

Angiogenesis Targeting

Angiogenesis Targeting

TTV 02 Angiogenesis Targeting: Mechanism of action animation that explains angiogenesis and it’s roll in cancer growth. VEGF factors in anti-angiogenic activity, helping to stop and reverse cancer growth and proliferation. Angiogenesis can be targeted by various types of drug agents (EGFR or HER1, VEGF, VEGF receptor and MMP inhibitors) which can interrupt the growth of tumor vasculature. Storyboard, Illustration and Animation by Jennifer Fairman.

TTV 02: Angiogenesis Targeting from Fairman Studios on Vimeo.

Angiogenesis can be targeted by various types of drug agents (EGFR or HER1, VEGF, VEGF receptor and MMP inhibitors) which can interrupt the growth of tumor vasculature. Storyboard, Illustration and Animation by Jennifer Fairman.

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