Our story begins in the 1980’s, an era defined by big hair, shoulder pads, and Miami Vice, and an era whose closing days would see a rather curious, unassuming discovery. Around this time, scientists found that sections of bacterial genomes contained repetitive sequences with no readily apparent function. These patterns contained palindromic repeats separated by clusters of 30 or so assorted bases termed “spacer DNA”. Researchers learned decades later that these spacer regions contained bits of viral DNA. The tipping point for this peculiar case came seven years ago when a team at Danisco discovered that by exposing S. thermophilus to bacteriophage DNA, they could alter the phage resistance of the bacteria. In the years following, a rather astounding story would unravel, one spearheaded by UC Berkeley’s own Dr. Jennifer Doudna and recently named a top contender (for the second year in a row) for Science magazine’s Breakthrough of the Year 2013.
As you, dear reader, have indubitably surmised from the title, the story above alludes to the tale of CRISPR. More formally known as “Clustered Regularly Interspaced Short Palindromic Repeats,” CRISPR is a novel genome editing technique based on the inherited, adaptable immune system of bacteria which has become red hot in the past two years (and a favorite topic of the BSR!). The usefulness of CRISPR in gene-editing can largely be attributed to the engineering feat of Doudna and her team, whose work took a place front and center following the 2007 findings at Danisco. At this time, her research was focused on developing an understanding of the role that the DNA spacers played in the bacterial immune system. In collaboration with Dr. Emmanuelle Charpentier at Umeå, rigorous study of the CRISPR-associated protein Cas9 began. Cas9 was found to be a nuclease, an enzyme that cuts DNA. When a bacterium is infected with a phage, it will cut up the viral DNA and insert these snippets into its own genome at CRISPR sites. This is then transcribed into RNA, eventually marking the viral genome for destruction. Doudna and Charpentier discovered that following transcription, RNA is processed into smaller functional segments (crRNAs). These crRNAs serve as recognition elements and, with the help of trans-activating crRNA (tracrRNA), complex with Cas9 to attack the viral DNA at specific locations determined by the RNA sequence. A few snips later, and the invader DNA is effectively silenced.
Here is where the team got clever; while Cas9 was demonstrated to assemble with two short RNA sequences (and thus possessed two active cutting sites), researchers showed that they could disable the cutting sites while maintaining the RNA-endowed specificity. If the RNA-enzyme complex could be simplified to work with one RNA strand instead of two, the genetic engineering potential would be enormous. Unlike other gene-editing techniques, like zinc fingers and TALENS, whereby a new enzyme needs to be made for every DNA sequence target, using an RNA-programmable nuclease would only require a new RNA sequence, and the enzyme would remain unchanged. This would not only be more simple and robust, but cheap.
Dr. Martin Jinek, then a postdoctorate in Doudna’s lab, was able to do just this. In 2012, he demonstrated that feeding Cas9 “single-guide RNA” could be used to target and cut areas of a genome with remarkable specificity. Overnight, the genetic engineering community exploded with what Science magazine called the “CRISPR Craze“. In the past year alone, this technique has been used to manipulate the genes in pretty much every organism important for biological research — mice, yeast, zebrafish, nematodes, drosophila, you name it. More recently, both Dr. George Church at Harvard and Doudna’s team have independently demonstrated that CRISPR can also be used on human cells to manipulate our DNA. In this instance, CRISPR was found to be more efficient and versatile when compared to TALENS. Several start-ups, including one co-founded by Doudna, have been formed around these techniques.
CRISPR technology may be more popular to scientists now than shoulder pads were in the 1980’s, but the technique still has a number of wrinkles to smooth out. Most pressing are concerns surrounding specificity and off-target mutagenesis. For a truly safe approach, CRISPR needs to accurately and precisely knock out genes. Delivery methods for gene therapy are also under consideration. However, given the flurry of activity surrounding this scientific gold mine, the future looks quite promising. Keep your eyes open.