Core Concept: CRISPR gene editing.

Just a few years ago, molecular biologists hoping to alter the genome of their favorite organisms faced an arduous task and likely weeks of genetic tinkering. Today, those scientists can quickly destroy or edit a gene with a new technology called CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9. “It really opens up the genome of virtually every organism that’s been sequenced to be edited and engineered,” says Jill Wildonger of the University of Wisconsin– Madison. CRISPR users have essentially coopted the immune system of bacteria, using the Cas9 enzyme to chop up the genomes of invading viruses. The trick is simple: the Cas9 enzyme cuts DNA at a specific sequence, determined by an accompanying bit of RNA called a guide RNA. Then, the cell’s own DNA repair machinery typically takes over in one of two different modes. In the first mode, it simply glues the two pieces back together, but imperfectly, so the leftover scar interrupts and disables the targeted gene. Or, in a second kind of repair, the cell can copy a nearby piece of DNA to fill in the missing sequence. By providing their own DNA template, scientists can induce the cell to fill in any desired sequence, from a small mutation to a whole new gene. The CRISPR/Cas9 genetic engineering system has become nearly ubiquitous in biology laboratories over the past few years. However, the original CRISPR researchers had no such application in mind, according to Jennifer Doudna of the University of California, Berkeley, one of the developers of the technology. Some of the first researchers to investigate CRISPR were food scientists at Danisco USA Inc.; they were worried about viruses infecting the Streptococcus strains used to make yogurt and cheese (1). These researchers observed that bacterial chromosomes contain oddly repetitive sequences called “clustered regularly interspaced short palindromic repeats,” or CRISPR. Between those repeats are sequences from viruses that infect bacteria. The microbes use the viral sequences as a mnemonic to remember past invaders. If the same virus should try to get a foothold again, the microbes are ready with an immune response that includes a copy of the remembered sequences, called a crRNA, and a second RNA, dubbed tracrRNA, encoded near the CRISPR repeats. Together, these RNAs recruit the Cas9 protein to viral DNA, and the enzyme cuts it up. CRISPRs were interesting mainly to microbiologists, Doudna points out, until 2012 when her group and that of collaborator Emmanuelle Charpentier, at Umeå University in Sweden, figured out they could combine crRNA and tracrRNA into a single, artificial guide RNA, which they could then use to aim the DNA-slicing enzyme at a sequence of their choosing (2). “We realized that we could use this for cutting DNA. . .in order to trigger repair,” Doudna recalls. The implications were thrilling. “I had chills going down my back,” she says. Doudna’s initial experiments were with molecules in test tubes, not living cells. But less than a year later, two other laboratories demonstrated the use of CRISPR/Cas9 to edit the genomes of cultured mouse and human cells in laboratory dishes (3, 4). Genetic engineers had already designed similar systems to snip DNA at any desired location, but they required scientists to assemble a protein to home in on every new target sequence, a tedious process. “Then along came CRISPR and, boom! You can just order an oligo[nucleotide] and make any change in the genome you wish,” says Dan Voytas, director of the Center for Genome Engineering at the University of Minnesota in Minneapolis, who developed one of the protein-based systems. Scientists can alter a gene—or several at once—and look for effects on their organism The use of the CRISPR-CAS9 gene-editing complex, illustrated here in Streptococcus pyogenes, has already had a major impact on multiple fields. Cas9 is shown in teal/blue, RNA in magenta and lime green. Image courtesy of Shutterstock/molekuul.be.