第 5 章
The CRISPR Revolution Begins
Roughly a year after their CRISPR article was published in Science, the scientific community had rapidly exploited their revelations, using CRISPR-Cas9 to engineer DNA in countless organisms and human cells. In what felt like the blink of an eye, Doudna had been transported from bacterial biology to the world of human medicine.
At Harvard, Professor Kiran Musunuru showed how his team was using CRISPR to develop treatments for genetic diseases like sickle cell disease. They had successfully used CRISPR to target and cut the mutated beta-globin gene, replacing the faulty letter A with the correct letter T-demonstrating CRISPR's potential to cure a disease affecting millions worldwide.
The approach could potentially be applied to many genetic diseases, but would require resources beyond what any academic laboratory could provide. In 2013, Doudna joined with George Church, Keith Joung, David Liu, and Feng Zhang to found Editas Medicine with $43 million in financing. Soon after, Emmanuelle Charpentier co-founded CRISPR Therapeutics, and Intellia Therapeutics joined the scene. By the end of 2015, these three companies would raise well over half a billion dollars to develop therapies targeting numerous disorders.
CRISPR quickly disseminated through the global scientific community as word spread that gene editing inside living cells could now be performed easily within days. In the days before CRISPR, gene editing required sophisticated protocols, formidable expertise, and substantial financial resources. Now, even laboratories with no prior gene-editing experience were using the technology.
As 2012 ended, Science magazine named genome editing a runner-up breakthrough of the year but highlighted TALENs rather than the newer CRISPR technology. Yet the first weeks of 2013 saw five additional CRISPR papers published, all demonstrating gene editing in cells as proposed. Papers from Feng Zhang, George Church, Jin-Soo Kim, Luciano Marraffini, and Keith Joung showed CRISPR editing over a dozen different genes in various cell types-from human leukemia cells to bacterial cells and even zebrafish embryos.
The excitement intensified in May 2013 when Rudolf Jaenisch's lab reported creating gene-edited mice using CRISPR. While previous methods required embryonic stem cells, extensive breeding, and many generations of mice-often an entire PhD thesis-Jaenisch's team achieved the same feat in just one month by microinjecting CRISPR components directly into one-cell embryos. Even more remarkably, they demonstrated one-step multiplex gene editing by using multiple RNA guides to edit several DNA sequences simultaneously.
By summer 2013, researchers began tracking all the different organisms whose genomes had been edited using CRISPR. The list grew rapidly-from zebrafish, bacteria, mice, and human cells to yeast, fruit flies, and worms. By late 2013, it included rats, frogs, and silkworms. By 2014, rabbits, pigs, goats, sea squirts, and monkeys had been added. Plant biologists soon demonstrated CRISPR's potential in crops like rice, sorghum, wheat, and later soybeans, tomatoes, oranges, and corn.
CRISPR is better described as a Swiss army knife than scissors, offering diverse functionalities from a single molecular machine. Its simplest use involves cutting a specific gene and allowing the cell to repair the damage by reconnecting the strands-a sloppy, error-prone process that leaves telltale insertions or deletions of DNA. These small changes disrupt the strict three-letter grouping of genetic code, causing the corresponding mRNA to produce wildly mutated proteins or, more commonly, no protein at all. This effectively destroys a gene's ability to function, a process geneticists call a "knockout."
Beyond simple knockouts, CRISPR enables precise genetic correction through homologous recombination. Unlike error-prone repair that merely rejoins broken DNA, this approach uses a repair template that matches the broken ends. This allows researchers to permanently replace harmful mutations with healthy sequences, making it possible to cure genetic diseases rather than merely disrupt genes.
Scientists discovered that deliberately disabling CRISPR's cutting ability creates a powerful tool for gene regulation. This "deactivated" CRISPR can still locate specific DNA sequences but acts as a molecular packhorse, carrying protein payloads that turn genes on or off like a dimmer switch. This approach offers more nuanced control than editing-comparable to fine-tuning instruments in an orchestra rather than replacing them entirely.
CRISPR's explosive popularity stems from its versatility, range, and accessibility. Unlike previous gene-editing tools that were expensive and technically demanding, CRISPR is affordable and user-friendly. The nonprofit organization Addgene has facilitated this democratization by distributing CRISPR plasmids to researchers worldwide for just $65 each, shipping some 60,000 CRISPR-related plasmids to over 80 countries in 2015 alone.
