Broad Institute-MIT team identifies highly efficient new Cas9 for in vivo genome editing

A collaborative study between researchers from the Broad Institute of MIT and Harvard, Massachusetts Institute of Technology, and the National Center for Biotechnology Information of the National Institutes of Health (NIH-NCBI) has identified a highly efficient Cas9 nuclease that overcomes one of the primary challenges to in vivo genome editing. This finding, published today in Nature, is expected to help make the CRISPR toolbox accessible for in vivo experimental and therapeutic applications.

Originally discovered in bacteria, the CRISPR-Cas9 system enables the cutting of DNA as a defense mechanism against viral infection. Although numerous microbial species possess this system, the Cas9 enzyme from Streptococcus pyogenes (SpCas9) was the first to be engineered for altering the DNA of higher organisms, and has since emerged as the basis for a series of highly versatile genome modification technologies.

Smaller packaging

In order to perturb genes in adult animals, key components of the CRISPR-Cas9 system must be introduced into cells using delivery vehicles known as vectors. Adeno-associated virus (AAV) is considered one of the most promising candidate vectors, as it is not known to cause human disease and has already gained clinical regulatory approval in Europe. However, the small cargo capacity of AAV makes it challenging to package both the SpCas9 enzyme and the other components required for gene editing into a single viral particle.

The Cas9 nuclease from the bacteria Staphylococcus aureus (SaCas9), presented in this new work, is 25% smaller than SpCas9, offering a solution to the AAV packaging problem.

The Broad/MIT team, led by Feng Zhang, core member of the Broad Institute and investigator at the McGovern Institute for Brain Research at MIT, along with collaborators at MIT, led by MIT Institute Professor Phillip Sharp, and the NCBI led by Eugene Koonin, set out to identify smaller Cas9 enzymes that could replicate the efficiency of the current SpCas9 system, while allowing packaging into delivery vehicles such as AAV. The researchers began by using comparative genomics to analyze Cas9s from more than 600 different types of bacteria, selecting six smaller enzymes for further study.

“Sifting through the 600 or so available Cas9 sequences, we identified a group of small variants in which the enzymatic domains were intact whereas the non-enzymatic portion was substantially truncated,” said Eugene Koonin, senior investigator with the NCBI and a contributing author of the study. “Luckily, one of these smaller Cas9 proteins turned out to be suitable for the development of the methodology described in this paper. We are now actively exploring the diversity of Cas9 proteins and their relatives in the hope to find new varieties that could potentially lead to even more powerful tools.”

After rigorous testing, only the Cas9 from S. aureus demonstrated DNA cutting efficiency comparable to that of SpCas9 in mammalian cells. The team then used a method known as BLESS, previously developed by Nicola Crosetto of the Karolinska Institute and Ivan Dikic at the Goethe University Medical School, to determine the presence of unintended “off-targets” across the entire genomic space. Again, SaCas9 and SpCas9 demonstrated comparable DNA targeting accuracy.

The team demonstrated the power of in vivo gene editing with AAV/SaCas9-mediated targeting of PCSK9, a promising drug target. The loss of PCSK9 in humans has been associated with the reduced risk of cardiovascular disease and lower levels of LDL cholesterol. In a mouse model, the team observed almost complete depletion of PCKS9 in the blood one week after administration of AAV/SaCas9 and a 40% decrease in total cholesterol. The mice showed no overt signs of inflammation or immune response.

“While we have chosen a therapeutically relevant target, PCSK9, in this proof-of-principle study, the greater goal here is the development of a versatile and efficient system that expands our ability to edit genomes in vivo,” said Fei Ann Ran, co-first author of the study, along with Le Cong and Winston Yan.

More broadly, SaCas9 is expected improve scientists’ ability to screen for the effects of mutations and better understand gene function using animal models. In the future, it may also be engineered to allow the targeted control of gene expression, which can be employed to expand our understanding of transcriptional and epigenetic regulation in the cell.

The next step, says senior author Feng Zhang, is to compare and contrast the two Cas9s in the hope of recognizing ways to further optimize the system.

“This study highlights the power of using comparative genome analysis to expand the CRISPR-Cas9 toolbox,” said Zhang. “Our long-term goal is to develop CRISPR as a therapeutic platform. This new Cas9 provides a scaffold to expand our Cas9 repertoire, and help us create better models of disease, identify mechanisms, and develop new treatments.”

About the engineered CRISPR-Cas9 system

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) have recently been harnessed as genome editing tools in a wide range of species. The engineered CRISPR-Cas9 system allows researchers to mutate or change the expression of genes in living cells, including those of humans. The family of Cas9 nucleases (also known as Cas5, Csn1, or Csx12) recognizes DNA targets in complex with RNA guides. Researchers can now harness the engineered system to home in on specific nucleic acid sequences and cut the DNA at those precise targets. The cuts modify the activity of the targeted genes, allowing researchers to study the genes’ function.

Broad, MIT researchers reveal structure of key CRISPR complex

Researchers from the Broad Institute and MIT have teamed up with colleagues from the University of Tokyo to form the first high definition picture of the Cas9 complex – a key part of the CRISPR-Cas system used by scientists as a genome-editing tool to silence genes and probe the biology of cells. Their findings, which are reported this week in Cell, are expected to help researchers refine and further engineer the tool to accelerate genomic research and bring the technology closer to use in the treatment of human genetic disease.

First discovered in bacteria in 1987, CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) have recently been harnessed as so-called genome editing tools. These tools allow researchers to home in on “typos” within the three-billion-letter sequence of the human genome, and cut out and even alter the problematic sequence. The Cas9 complex, which includes the CRISPR “cleaving” enzyme Cas9 and an RNA “guide” that leads the enzyme to its DNA target, is key to this process.

“We’ve come to view the Cas9 complex as the ultimate guided missile that we can use to target precise sites in the genome,” said co-senior author Feng Zhang, a core member of the Broad Institute, an investigator at the McGovern Institute for Brain Research, and an assistant professor at MIT. “This study provides a schematic of the entire system – it shows the missile (the Cas9 protein), the programming instructions (the guide RNA) that send it to the right location, and the target DNA. It also reveals the secret of how these pieces function together to make the whole system work.”

To deconstruct this system, Zhang approached the paper’s co-senior author Osamu Nureki at the University of Tokyo. Together, they assembled a team to work out the complicated structure.

“Cas9-based genome editing technologies are proving to be revolutionary in a wide range of life sciences, enabling many new experimental techniques, so my colleagues and I were excited to work with Feng’s lab on this important research,” said first author Hiroshi Nishimasu, an assistant professor of biophysics and biochemistry who works in Nureki’s lab at the University of Tokyo.

The two teams worked closely to reveal the structural details of the Cas9 complex and to test their functional significance. Their efforts revealed a division of labor within the Cas9 complex. The researchers determined that the Cas9 protein consists of two lobes: one lobe is involved in the recognition of the RNA and DNA elements, while the other lobe is responsible for cleaving the target DNA, causing what is known as a “double strand break” that disables the targeted gene. The team also found that key structures on Cas9 interface with the guide RNA, allowing Cas9 to organize itself around the RNA and the target DNA as it prepares to cut the strands.

Identifying the key features of the Cas9 complex should enable researchers to improve the genome-editing tool to better suit their needs.

“Up until now, it has been very difficult to rationally engineer Cas9. Now that we have this structural information, we can take a principled approach to engineering the protein to make it more effective,” said Zhang, who is also a co-founder of Editas Medicine, a company that was started last year to develop Cas9 and other genome editing technologies into a novel class of human therapeutics.

Currently, Cas9 is used in experiments to silence genes in mammalian cells – sometimes at multiple sites across the genome – and large libraries of RNA sequences have been created to guide Cas9 to genes of interest. However, the system can only target specific types of sites. Some studies have also shown that the RNA could lead Cas9 “off-target,” potentially causing unexpected problems within the cellular machinery.

The researchers plan to use this new, detailed picture of the Cas9 complex to address these concerns.

“Understanding this structure may help us engineer around the current limitations of the Cas9 complex,” said study author F. Ann Ran, a graduate student in Zhang’s lab. “In the future, it could allow us to design versions of these editing tools that are more specific to our research needs. We may even be able to alter the type of nucleic acid sequences that Cas9 can target.”

Such technological improvements will be needed if the CRISPR-Cas system is to evolve into a therapeutic tool for the treatment of genetic disease.

The study was supported by the National Institute of Mental Health (NIMH); an NIH Director’s Pioneer Award; the Japan Science and Technology Agency; the Japan Society for the Promotion of Science; the Keck, McKnight, Poitras, Merkin, Vallee, Damon Runyon, Searle Scholars, Klingenstein, and Simons Foundations; as well as Bob Metcalfe and Jane Pauley.

Other researchers who worked on the study include Patrick D. Hsu, Silvana Konermann, Soraya Shehata, Naoshi Dohmae, and Ryuichiro Ishitani.

Written by Veronica Meade-Kelly, Broad Institute

Paper cited:

Nishimasu H et al. “Crystal structure of Cas9 in complex with guide RNA and target DNA.” Cell DOI: 10.1016/j.cell.2014.02.001

About the Broad Institute of Harvard and MIT
The Eli and Edythe L. Broad Institute of Harvard and MIT was launched in 2004 to empower this generation of creative scientists to transform medicine. The Broad Institute seeks to describe all the molecular components of life and their connections; discover the molecular basis of major human diseases; develop effective new approaches to diagnostics and therapeutics; and disseminate discoveries, tools, methods and data openly to the entire scientific community.

Founded by MIT, Harvard and its affiliated hospitals, and the visionary Los Angeles philanthropists Eli and Edythe L. Broad, the Broad Institute includes faculty, professional staff and students from throughout the MIT and Harvard biomedical research communities and beyond, with collaborations spanning over a hundred private and public institutions in more than 40 countries worldwide. For further information about the Broad Institute, go to

About the McGovern Institute for Brain Research at MIT
The McGovern Institute for Brain Research at MIT is led by a team of world-renowned neuroscientists committed to meeting two great challenges of modern science: understanding how the brain works and discovering new ways to prevent or treat brain disorders. The McGovern Institute was established in 2000 by Patrick J. McGovern and Lore Harp McGovern, who are committed to improving human welfare, communication and understanding through their support for neuroscience research. The director is Robert Desimone, formerly the head of intramural research at the National Institute of Mental Health.