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A tentacled bacteriophage floats to a bacterial cell and robotically inserts its DNA. The cell soon starts making foreign proteins and turning them into new bacteriophages, which take over the cell and eventually destroy it.
Sometimes, though, the bacterium survives and remembers its invader. As a result, the next time the phage comes, the cell deftly destroys the foreign DNA. Though just a simple cell, the bacterium’s adaptive immunity is surprisingly sophisticated.
Over the past two years, scientists have begun taking advantage of this bacterial immunity, called CRISPR, to target genes in all sorts of cells.
“CRISPR-Cas9 has triggered a revolution in which laboratories around the world are using the technology for innovative applications in biology,” raves one recent review in Science (1).
Now a new paper in Cell reports an engineered CRISPR system that can repress or activate multiple genes at a time, easily and inexpensively (2). Study of complex, polygenic diseases (many of which plague the Western world) may never be the same.
The Influence of Yogurt
The story begins almost 30 years ago. In 1987, scientists discovered something strange inside the E. coli genome: short repeating elements interspersed with variable DNA sequences dubbed CRISPRs (clustered regularly interspaced palindromic repeats) (3). Soon, CRISPRs were found in other bacteria and archaea, but for a long time no one knew why they were there or what they did. Finally, in 2005, several papers showed that these 20–30 base pair snippets of DNA in bacteria often matched foreign bits of DNA from enemy bacteriophages or plasmids and that a neighboring gene, Cas9 (CRISPR-associated), codes for a protein with nuclease domains. Researchers began hypothesizing that CRISPR-Cas9 might play a role in protecting bacteria from attack by bacteriophages or other foreign elements.
In 2007, a serendipitous convergence of science and the food industry—specifically, a yogurt company—confirmed the theory (4). To make yogurt, companies add bacteria such as Streptococcus thermophilus to the milk. Unfortunately, parasitic bacteriophages often infect these cultures, ruining entire yogurt batches. Dairy scientists are always looking for bacterial cultures that are immune to bacteriophages, and at Danisco (now owned by DuPont), researchers started studying whether CRISPR could protect bacteria from phages. The result was a pivotal paper in Science showing that CRISPR provides acquired resistance to bacteriophages.
When a bacteriophage attacks a bacterium, the bacterium sometimes adds a piece of that phage’s DNA to its CRISPR region, like a memory pearl on a string. The snippet is now ready to be transcribed into guide RNA and attached to Cas9, where it guides binding of foreign DNA via Watson-Crick base pairing and allows the Cas9 endonuclease to destroy it.
In 2012, research teams started engineering the CRISPR-Cas9 system to target pieces of DNA in vitro (5). By creating their own guide RNA attached to Cas9, they could target any gene to induce a double-stranded break. After this work was published in Science, the field exploded, with teams editing genes in various cell types, including human cells, studying gene function, and so on. More than 1000 papers have been published describing the use of CRISPR, most of them since early 2013.
But one research group wasn’t interested in altering genes using CRISPR, they wanted to regulate them by activating and repressing sets of genes.
Working in Groups
In February 2013, Lei S. Qi, then at the University of California, San Francisco (UCSF), and Wendell Lim, also at UCSF, demonstrated in a paper in Cell that CRISPR could reversibly inhibit genes (6). First, Qi and his team turned off one of Cas9’s basic functions, the ability to cut double-stranded DNA, creating a “dead” Cas9 (dCas9), which lacked endonuclease activity, and linked it to normal guide RNA. This new DNA recognition complex could block transcription of particular genes without altering the genetic code. The researchers called their new system CRISPR interference (CRISPRi). Later that year, Qi’s group published another paper in Cell showing that they could also activate transcription of targeted genes using engineered guide RNA in human and yeast cells by coupling dCas9 to an activator (7).
Previously, Qi’s group used a CRISPR-dCas9 complex that contained a 20-nucleotide DNA-targeting sequence and a pair of structured RNA domains. Now the team has created a scaffold, adding an RNA hairpin domain and protein-recruiting RNA sequences that can attract activators or repressors. The group showed activation of some genes and repression of others in a synthetic multigene transcriptional program in yeast and human cells, expressing sets of enzymes in various combinations to lead to different outputs.
“Moving beyond controlling the single gene, we can control multiple genes differently. You can really use this tool for multiplex control of genes as a network,” said Qi. “On the other biomedical research side, it’s also opened up the possibility to study multi-gene networks because, especially for polygenic diseases, you really need to study how these genes work together to cause a phenotype and then use these tools to control many genes.”
Most common diseases are polygenic, with multiple genes turning on and off to create the phenotype. “This tool allows one to very flexibly control several genes and understand the interplay with expression of these genes…that will provide a much bigger space to treat complex disease and study how affecting multiple genes can bring the cell back to normal,” said Qi.
1. Doudna, J.A. et al, “The new frontier of genome engineering with CRISPR-Cas9,” Science, 28 November 2014.
2. Zalatan, J.G. et al, “Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds,” Cell, 15 January 2015.
3. Ishino, Y. et al, “Nucletotide sequence of the yap gene, responsible for alkaline phosphatase isozyme conversion in E. coli, and identification of the gene product,” J. Bacteriol, 1987.
4. Jinek, M et al, “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity,” Science, 17 August 2012.
5. Jinek, M et al, “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity,” Science, 17 August 2012.
6. Qi, L.S. et al, “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression,” Cell, 28 February 2013.
7. Gilbert, L.A. et al, “CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes,” Cell, 18 July 2013.
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