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New CRISPR/Cpf1 Genome Editing System Makes Waves at CSHL Meeting

This story has been changed to correct the year in which Charpentier and Doudna introduced Cas9 as an RNA-guided endonuclease.

NEW YORK (GenomeWeb) – Feng Zhang's opening night presentation at last week's Cold Spring Harbor Laboratory meeting on genome engineering was deceptively titled "Genome Editing Using CRISPR-Cas9." The scientists in attendance Thursday evening could have been forgiven for expecting a generic, introductory presentation or at the very least a presentation about Cas9.

Zhang's co-chairperson for the session, CRISPR/Cas9 pioneer Emmanuelle Charpentier, had kicked off the session with an account of how the technology had gone from bacterial curiosity to the driving force in the field of genome editing. Speaking in the middle of the session, Zhang, of the Broad Institute and the Massachusetts Institute of Technology, then proceeded to show the field a new direction it could move in.

"We were all sitting in this room expecting some sort of introductory talk," David Courtney, a post-doc from Ulster University who had travelled from the UK for the meeting, told GenomeWeb. "He blew us all out of the water. It was like Steve Jobs giving an Apple keynote presentation."

Zhang barely mentioned Cas9. Instead, he showed off an entirely new CRISPR-based, RNA-guided genome editing system that his lab had discovered, based on a protein called Cpf1. He published a study detailing the protein the next day in Cell.

CRISPR/Cpf1 editing works comparably well to CRISPR/Cas9, Zhang said in his talk, and has several exploitable differences that could make it an important addition to the gene-editing toolkit: it's smaller than Cas9, only uses one RNA strand to find its genomic target, and recognizes a different protospacer adjacent motif (PAM) sequence — which opens up new editing sites.

One attendee marveled at the bar the study set — taking what was a mostly unknown protein and turning it into a tool that increased the available targets in genomes of many organisms and could help move CRISPR towards the clinic. Still, some scientists were cautious, suggesting Cpf1 would not topple Cas9 as the protein of choice.

In some sense, Zhang's work is not surprising, since his lab has been an epicenter of innovation in the CRISPR/Cas9 revolution. Cas9 from Streptococcus pyogenes, known as spCas9, has been the gold-standard for CRISPR-based genome editing ever since Charpentier and Jennifer Doudna introduced that enzyme as an RNA-guided endonuclease in 2012. Zhang's lab has done engineering work to expand spCas9's capabilities, demonstrating multiplexed editing and the ability to increase specificity using two single-stranded nicks instead of one double-stranded break. Zhang has also led work on characterizing Cas9 enzymes from other bacterial species, introducing an enzyme from Staphylococcus aureus, or saCas9, that is smaller than spCas9 and thus easier to deliver to cells in vivo. Zhang currently holds several US Patents on CRISPR/Cas9 RNA-guided genome editing technology, though ownership of the intellectual property is currently disputed.

Moreover, CRISPR/Cas9 is just one type of bacterial immune system found in nature. "We've known for awhile that there are these other Cas9-like proteins in the CRISPR world," said Martin Jinek, a professor at the University of Zurich. Jinek worked as a post-doc in Doudna's laboratory at the University of California, Berkeley and was instrumental in developing CRISPR/Cas9 as a research tool. Scientists have been working to characterize them, but even those at the forefront of the field seemed surprised by Zhang's announcement.

"I didn't know he was so far ahead," Jinek said.

At one point, it looked as though Cpf1 might not be able to edit human cells. Rather than give up, Zhang and his colleagues tried more than a dozen Cpf1 orthologs and eventually found two that did work in human cells.

The lab found Cpf1 by looking through genomic databases for sequences similar to those of Cas9 proteins. Cpf1 is predicted to have a Cas9-like RuvC endonuclease domain, but has several key differences. Rather than the blunt cut of Cas9, Cpf1 leaves an overhang on one end of DNA. It also only needs one RNA to find its target (Cas9 requires two, but in practice they have been engineered into a single guide RNA). The significance of these quirks isn't clear, but Zhang suggested they could somehow be exploited to make genome editing more efficient.

The Cpf1 protein is also smaller than Cas9 —which measures over 4 kilobases, thus it's theoretically easier to deliver in clinical applications. Since clinical use, even with the well-characterized Cas9 enzyme, is at least several years away, Cpf1's initial value will likely come as a research tool. While spCas9 works well in many different systems and allows researchers to target many sites in the genome, its use is somewhat limited by its PAM, a triad of base pairs that serve as a landing site for the RNA-protein complex.

SpCas9 has a simple NGG PAM, which arises frequently in many genomes, approximately every 15 bases. But some genomic regions are more AT-rich, making them hard to target with spCas9. "Who wants to be looking for two Gs in a row when you could be looking for anything you want?" Nancy Maizels, a professor at the University of Washington, said. "He magnified the universe."

Some organisms have entire genomes that are AT-rich, like that of Plasmodium falciparum, which causes malaria. According to Gabrielle Josling, a post-doc at Pennsylvania State University, as much as 80 percent of the P. falciparum's genome is AT-dominated.

With a PAM favoring Ts over Gs, Cpf1 could solve a lot of problems Josling has faced in getting CRISPR genome editing to work. "We're super excited about it. It could be a game changer for us," she said.

Just the week before, she had attended the Molecular Parasitology Meeting at Woods Hole, Massachusetts. "Everyone was talking about how we needed a new Cas9," for genome editing, she said, one that was more amenable to the P. falciparum genome.

The Cell paper has already made an impression in her field, she said. By Saturday, she'd received half a dozen emails about CRISPR/Cpf1. "In Plasmodium I can imagine it will be readily adopted," she said.

But not all researchers will be so eager to move away from CRISPR/Cas9, predicted Samira Kiani, a post-doc at MIT and a synthetic biologist who has used the tool to engineer genetic circuits. "I don't think the field will be receptive for a whole new system right now," she said.

It's only been around for three years, but the field has moved so far and so fast that people are comfortable using Cas9. Besides, CRISPR/Cas9 works very well for lots of applications. "It will take a while before people adopt another system," she said.