A new method for more easily and precisely engineering the human genome may help scientists pin subtle changes in DNA to disease, moving the needle from correlation to causality and potentially improving gene therapy techniques.
Two groups recently reported in Science their success using a tool borrowed from a bacterial immune system called Cas, short for CRISPR-associated systems, which in turn stands for Clustered Regularly Interspaced Short Palindromic Repeats. In bacteria the Cas9 enzyme system uses short stretches of RNA to target and then cut invading viral DNA. Scientists have customized this system to work in human cells, creating an RNA-guided editing tool that allows them to integrate DNA changes into the genomes of living cells, a process called genome engineering.
One of the teams, led by George Church, HMS Robert Winthrop Professor of Genetics, also tested the method in induced pluripotent stem cells (iPSCs), an important milestone on the path to human genome engineering. These cells, taken from a child or adult, have been modified to mimic embryonic cells, which means they can develop into any adult cell type. In experiments, iPSCs offer greater clarity than traditional cell lines as researchers explore gene function in different cell types.
The other team, led by Feng Zhang of the Broad Institute of Harvard and MIT, independently showed the effectiveness of Cas9.
While the Cas9 method is still a long way from the clinic, Church is hopeful.
“We need a lot more experience and optimization, but it looks very promising,” he said. “This is much easier than any previous human genome engineering method, and it is relevant to testing the flow of ideas from GWAS [genome-wide association studies] and the Personal Genome Project.”
The Cas9 approach, first developed by Harvard University graduate Jennifer Doudna of the University of California, Berkeley, could end up supplanting a technique that Science just last month named one of the top 10 scientific breakthroughs of 2012. For this technique, a class of proteins called TALENs (Transcription Activator-Like Effector Nucleases) zero in on a particular region of the genome, where they can precisely cut DNA and insert or delete a gene. TALENs followed a method from the 1990s that used enzymes called zinc finger nucleases to target specific parts of genomes.
TALENs were easier to design, but, like zinc fingers, they require about 2,000 bases of messenger RNA to encode an enzyme that would cleave DNA at a specific site in a genome. Cas9 needs 1/100 as much RNA; as few as 20 variable bases, embedded in short constant guide RNAs, are sufficient for precise targeting, the scientists showed in human cell lines.
According to Church, also testing Cas9 in iPSCs is particularly noteworthy.
“We need to have human stem cells in culture so we can manipulate the genome and see how the cells differentiate, or fit into tissues,” he said. “To change from correlation and speculation to causality, you introduce single changes, one by one, into a test genome and ask which of those or how many of those do you need in order to see the trait. That turns correlation into causation and moves it closer to the gold standard.”
The more compact Cas9 system opens the door to engineering multiple changes in different genes and then testing them simultaneously to see what role they play in complex diseases. Large genome-sequencing studies can find a variety of genes active in people with a particular disease, but it takes the kind of multiplex testing Cas9 allows to establish which ones actually matter, Church said.
A decade ago early attempts at gene therapy failed because delivery of a new, corrected gene could inadvertently promote cancer or provoke a harmful immune system response. Newer methods, in addition to having more precise delivery, are also designed to have lower toxicity and generate only a tolerable immune response in the case of a rare off-target event. These newer approaches also make it possible to target an old gene with new information, either knocking it out or changing it precisely.
Cas9 has the potential to accelerate these improvements in gene therapy, but much more testing is necessary to see if it will continue to display the greater efficiency and very low toxicity shown in these early experiments. “But it looks like a very promising starting point,” Church said.
This work was supported by NIH grant P50 HG005550. Church and the Science paper’s first author, research fellow Prashant Mali, have applied for a patent based on the findings of this study.