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Snip vs. Shred

While CRISPR-Cas9 genome editing gets all the headlines, its cousin may revolutionize the fight against bad bacteria.

Story by Alexander Gelfand • Illustrations by  Jennifer E. Fairman, CMI, FAMI

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 bacteriophages (viruses that infect bacteria). 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 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.

SNP FPO

Cas9 acts as both an antibody and an enzyme, giving bacteria the power to recognize viral DNA.

Snip Step 1

Virus binds to bacterial cell membrane

Snip Step 2

Viral DNA enters bacterial cell cytosol

Snip Step 3

Cas9 protein recognizes viral DNA with guide RNA

Target DNA sequence wrapped around guide RNA

Snip Step 4

Precise Cuts
The Cas9 protein cuts neatly through viral DNA like a pair of molecular scissors

SNP FPO

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 particle accelerators at Stanford University and Brookhaven National Laboratory.

Shred Step 1

Virus binds to bacterial cell membrane

Shred Step 2

Viral DNA enters bacterial cell cytosol

Shred Step 3

Cascade protein recognizes viral DNA with guide RNA

Target DNA sequence unwinds and flattens into a ladder-shaped configuration

Shred Step 4

Cascade recruits the Cas3 protein to bind to viral DNA


Cas3 has enzymatic properties that unzip and shred viral DNA.

Shred Step 5

Cas3 unzips viral DNA double-helix into single strands

Shred Step 6

DNA Shredder
Cas3, employed by the CRISPR-Cascade, chews viral DNA up like a shredder

Bacteria use CRISPR-Cas systems to store the genetic signatures of phages that have previously infected them. These viral mug shots appear in host DNA as stretches of viral DNA separated by short, repeated sequences. (CRISPR stands for “clustered, regularly interspaced, short palindromic repeats.”) When a phage invades, CRISPR-Cas compares phage DNA to its archive of previous invaders. If a match is found, Cas9 cuts the invading DNA like a pair of molecular scissors, while Cascade employs Cas3 to shred the viral DNA.

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.

Milk Carton

Origins

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.

DNA Strand

Applications

CRISPR-Cas9 has been used to edit genes in wheat, trees, monkeys and mice. In the lab, researchers have used it to remove HIV DNA from a human genome and to fix a mutation that causes cystic fibrosis.

Chemistry Flask

Insights

Understanding CRISPR- Cascade would help the pharmaceutical industry, which uses genetically engineered bacteria and yeast to produce a variety of drugs. It also could help scientists weaken the bacterial immune system to kill harmful microbes, prevent antibiotic resistance and deliver new ways of grappling with bacteria.

 

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