Scientists on the McGovern Institute for Brain Research at MIT and the Broad Institute of MIT and Harvard have re-engineered a compact RNA-guided enzyme they present in bacteria into an efficient, programmable editor of human DNA.
The protein they created, called NovaIscB, might be adapted to make precise changes to the genetic code, modulate the activity of specific genes, or perform other editing tasks. Because its small size simplifies delivery to cells, NovaIscB’s developers say it’s a promising candidate for developing gene therapies to treat or prevent disease.
The study was led by Feng Zhang, the James and Patricia Poitras Professor of Neuroscience at MIT who can be an investigator on the McGovern Institute and the Howard Hughes Medical Institute, and a core member of the Broad Institute. Zhang and his team reported their open-access work this month within the journal .
NovaIscB is derived from a bacterial DNA cutter that belongs to a family of proteins called IscBs, which Zhang’s lab discovered in 2021. IscBs are a sort of OMEGA system, the evolutionary ancestors to Cas9, which is a component of the bacterial CRISPR system that Zhang and others have developed into powerful genome-editing tools. Like Cas9, IscB enzymes cut DNA at sites specified by an RNA guide. By reprogramming that guide, researchers can redirect the enzymes to focus on sequences of their selecting.
IscBs had caught the team’s attention not only because they share key features of CRISPR’s DNA-cutting Cas9, but additionally because they’re a 3rd of its size. That will be a bonus for potential gene therapies: compact tools are easier to deliver to cells, and with a small enzyme, researchers would have more flexibility to tinker, potentially adding latest functionalities without creating tools that were too bulky for clinical use.
From their initial studies of IscBs, researchers in Zhang’s lab knew that some family members could cut DNA targets in human cells. Not one of the bacterial proteins worked well enough to be deployed therapeutically, nonetheless: the team would have to switch an IscB to make sure it could edit targets in human cells efficiently without disturbing the remainder of the genome.
To start that engineering process, Soumya Kannan, a graduate student in Zhang’s lab who’s now a junior fellow on the Harvard Society of Fellows, and postdoc Shiyou Zhu first looked for an IscB that may make good place to begin. They tested nearly 400 different IscB enzymes that might be present in bacteria. Ten were able to editing DNA in human cells.
Even essentially the most lively of those would must be enhanced to make it a useful genome editing tool. The challenge can be increasing the enzyme’s activity, but only on the sequences specified by its RNA guide. If the enzyme became more lively, but indiscriminately so, it might cut DNA in unintended places. “The bottom line is to balance the advance of each activity and specificity at the identical time,” explains Zhu.
Zhu notes that bacterial IscBs are directed to their goal sequences by relatively short RNA guides, which makes it difficult to limit the enzyme’s activity to a particular a part of the genome. If an IscB may very well be engineered to accommodate an extended guide, it might be less more likely to act on sequences beyond its intended goal.
To optimize IscB for human genome editing, the team leveraged information that graduate student Han Altae-Tran, who’s now a postdoc on the University of Washington, had learned in regards to the diversity of bacterial IscBs and the way they evolved. As an example, the researchers noted that IscBs that worked in human cells included a segment they called REC, which was absent in other IscBs. They suspected the enzyme might need that segment to interact with the DNA in human cells. Once they took a more in-depth take a look at the region, structural modeling suggested that by barely expanding a part of the protein, REC may also enable IscBs to acknowledge longer RNA guides.
Based on these observations, the team experimented with swapping in parts of REC domains from different IscBs and Cas9s, evaluating how each change impacted the protein’s function. Guided by their understanding of how IscBs and Cas9s interact with each DNA and their RNA guides, the researchers made additional changes, aiming to optimize each efficiency and specificity.
In the long run, they generated a protein they called NovaIscB, which was over 100 times more lively in human cells than the IscB that they had began with, and that had demonstrated good specificity for its targets.
Kannan and Zhu constructed and screened tons of of recent IscBs before arriving at NovaIscB — and each change they made to the unique protein was strategic. Their efforts were guided by their team’s knowledge of IscBs’s natural evolution, in addition to predictions of how each alteration would impact the protein’s structure, made using a man-made intelligence tool called AlphaFold2. In comparison with traditional methods of introducing random changes right into a protein and screening for his or her effects, this rational engineering approach greatly accelerated the team’s ability to discover a protein with the features they were on the lookout for.
The team demonstrated that NovaIscB is an excellent scaffold for quite a lot of genome editing tools. “It biochemically functions very similarly to Cas9, and that makes it easy to port over tools that were already optimized with the Cas9 scaffold,” Kannan says. With different modifications, the researchers used NovaIscB to interchange specific letters of the DNA code in human cells and to alter the activity of targeted genes.
Importantly, the NovaIscB-based tools are compact enough to be easily packaged inside a single adeno-associated virus (AAV) — the vector mostly used to securely deliver gene therapy to patients. Because they’re bulkier, tools developed using Cas9 can require a more complicated delivery strategy.
Demonstrating NovaIscB’s potential for therapeutic use, Zhang’s team created a tool called OMEGAoff that adds chemical markers to DNA to dial down the activity of specific genes. They programmed OMEGAoff to repress a gene involved in cholesterol regulation, then used AAV to deliver the system to the livers of mice, resulting in lasting reductions in levels of cholesterol within the animals’ blood.
The team expects that NovaIscB might be used to focus on genome editing tools to most human genes, and stay up for seeing how other labs deploy the brand new technology. In addition they hope others will adopt their evolution-guided approach to rational protein engineering. “Nature has such diversity, and its systems have different benefits and drawbacks,” Zhu says. “By learning about that natural diversity, we will make the systems we try to engineer higher and higher.”
This study was funded, partly, by the K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics at MIT, Broad Institute Programmable Therapeutics Gift Donors, Pershing Square Foundation, William Ackman, Neri Oxman, the Phillips family, and J. and P. Poitras.
