Skip to main content
Premium Trial:

Request an Annual Quote

Agilent, Stanford Researchers Collaborate on Chemically Modified Guide RNAs for CRISPR/Cas9 Genome Editing in T Cells


NEW YORK (GenomeWeb) – A collaboration between researchers at Stanford University and Agilent Technologies may have unlocked CRISPR/Cas9 genome editing in T cells and other primary cells by chemically modifying the guide RNA (gRNA) that lead the Cas9 nuclease to its genomic target.

While researchers have been extremely successful in getting CRISPR/Cas9 genome editing to work in a number of different model organisms and human cell types, they were not only having trouble using it in primary cells — including T cells and hematopoietic stem cells — they couldn't get it to work at all.

It wasn't for lack of trying; several labs were working on the problem, Matthew Porteus, the primary investigator of the Stanford lab involved in the collaboration, told GenomeWeb. But the scientists didn't even know why it wasn't working. "Nobody was talking about it because no one wanted to admit they couldn't get it to work," he said.

Now, the Stanford and Agilent scientists have gotten CRISPR/Cas9 to work in those types of cells, by synthesizing guide RNAs with added chemical modifications and delivering them alongside a Cas9 mRNA or even with the protein as an assembled whole, rather than transcribing it off a plasmid.

The two groups of scientists published some results of their collaborative study detailing different levels of protection against exonuclease degradation by the cell the modifications provide. They published the study in June in Nature Biotechnology.

The collaboration married the genome editing expertise of Porteus' lab with the nucleic acid synthesis capabilities of Agilent, the scientists said, and the benefits cut both ways.

Porteus' lab, which has studied genome editing-based approaches to treating diseases such as thalassemia and sickle cell disease, now has an efficient and scalable platform for developing therapies.

"It was naturally of interest to us to explore how our expertise in nucleic acid synthesis could be advantageous for future customers using CRISPR technologies, Steve Laderman, director of Agilent Research Labs, told GenomeWeb.

Having developed the key technology to enable scientists to edit T cells with CRISPR/Cas9, future customers could include a host of pharmaceutical companies interested in developing immuno-oncology treatments.

"If you look at the literature, there was nothing saying it would work," Porteus said. His lab has been interested in genome engineering to treat genetic diseases "as long as anybody," he said. "We've used zinc finger nucleases and [transcription activator-like effector nucleases], we've been using CRISPR/Cas9. We had a pretty good flavor for the pros and cons of each platform."

Like other labs trying to edit genomes in primary cells, the scientists in Porteus' lab weren't having much success. "We were actually getting really low frequencies [of edits with CRISPR/Cas9], even lower than with other platforms," such as TALENs, he said.

Porteus said they had two prevailing theories as to why CRISPR/Cas9 wasn't working in primary cells. One theory was that the DNA plasmids used to express the Cas9 enzyme and the gRNA were not designed properly to work in the cells, perhaps because the promoters used were not active enough. The other hypothesis was that delivering DNA into primary cells activated an innate immune response, that the cell viewed it as toxic.

"We don't know which is true," Porteus confessed, "but we do know that by delivering things as RNA, we solved the problem."

To get around delivering CRISPR/Cas9 as a DNA plasmid, Porteus' lab realized that they would need to synthesize the single gRNA, which can be at least 100 nucleotides long. Serendipitously, Stanford postdoc and first author on the Nature Biotechnology paper Ayal Hendel attended a talk given by a scientist from Agilent's research laboratories about the firm's proprietary RNA synthesis chemistry. Laurakay Bruhn, section manager for biological chemistries at Agilent Research Labs, told GenomeWeb that Agilent's chemistry can make RNA "significantly longer, in general, and more robustly than other methods." Moreover, Agilent "had already done a fair amount of work" looking at the topic area of synthesizing chemically modified gRNAs with its method.

Laderman explained that "a hundred-mer is well established as single-guide RNA length that does work quite well. Different syntheses have more or less capabilities to make hundred-mers. Ours is particularly efficient in coupling and can easily make lengths like that."

Simply purifying enough gRNA for the types of experiments Porteus and Hendel wanted to do was beyond the capabilities of most other synthesis methods, Bruhn said. "At that time, most chemistries were quite challenged to get to that length in a robust way with enough yield to purify them effectively. Having an RNA synthesis chemistry that's very scalable that can make amounts from micrograms to milligrams up to grams with the same chemistry is useful for people thinking about translating from research to the clinic."

It turned out that being able to chemically modify the gRNAs was important, too. "While delivering [unmodified] gRNA might work in certain circumstances, they were susceptible to nuclease degradation," Porteus said. "You needed to modify the ends of the gRNA to make them resistant to nucleases."

The Agilent scientists came up with three tiers of protection against exonucleases, but Hendel said that gRNA can also be endowed to to have other useful features. Other chemical modifications might make them fluoresce, affect their specificity, or help bring them into or out of the cells.

Bruhn stressed that making chemical modifications to RNA is not necessarily new territory. "People have been putting modifications into RNAs, especially [small interfering RNAs], which people have been working on for years and years as potential therapeutics," she said.  "There's a whole assortment of modification when doing chemical synthesis you can incorporate into RNA at any position."

With the guides in hand, in the short to medium term, Porteus would like to achieve high frequencies of gene editing to correct mutations underlying the diseases his lab studies. Ultimately, though, the goal is "to be able to take a patient's own cells, correct mutations, and give them back to the patient." Synthetic guides are a critical, if not essential, component to that process, he said.

What Porteus describes doing is essentially the same goal for autologous T cell therapies developed to treat hematological cancers.

Because of the promise of these therapies, synthetic guides could turn CRISPR/Cas9 into the gene editing platform of choice in that field. "I know that what we've shown has enabled people interested in that space to do the experiments and cell manipulations that they've wanted to do," Porteus said, though he declined to disclose who those scientists were, saying the results had been shared in confidence.

For its part, Agilent declined to disclose whether it has applied for intellectual property related to the chemically modified gRNAs.

"The project is still a research and development project inside our central research labs," Laderman said. "Certainly one of our goals is to help understand the opportunity so the relevant businesses could come to a decision as to whether or not that makes sense. We're exploring our ability to create something that would be desirable from a customer perspective, as an early step towards clarifying the opportunity for the business. Other factors would then have to be looked at to get to make decisions about commercial plans."