New CRISPR-Cas9 strategy edits genes 2 ways

A team of Harvard and MIT researchers have developed a way to perform genome engineering as well as gene regulation at the same time.

The CRISPR-Cas9 system came into the limelight in 2013 when Jennifer A. Doudna PhD at the University of California, Berkeley, made one of the most monumental discoveries in biology: an easy way to alter DNA. She created a gene editing tool that works similarly to how we edit words when writing a document: CRISP-Cas9. However, the first sweeping patents for the technology were granted not to her, but to Feng Zhang, a scientist at the Broad Institute and M.I.T. The University of California is challenging this decision, and a nasty skirmish still exists within the field.

CRISPR-Cas9 is still a revolutionary gene engineering tool. The Cas9 protein is found in the immune system of the streptococcus pyogene bacteria and acts like a pair of molecular scissors to precisely cut or edit specific sections of DNA. In the bacteria, cutting a viral gene and incorporating it into its own bacterial genome, nullifies that virus while building immunity.

More recently, however, scientists are combining use of CRISPR-Cas9 as a tool to regulate genes by turning genes off or on and through splicing.

Whether (1) splicing DNA or (2) turning genes off and on, genome engineering and regulation is initiated by a common step. The Cas9 protein targets specific genes on the DNA chain found by using a matched sequence of RNA guides that latch onto them. Until this latest innovation from the Wyss Institute collaboration with M.I.T., each of these two gene engineering feats required different variants of Cas9.

Splicing DNA depends on Cas9's innate ability to cleave DNA, but turning DNA on and off was achieved by removing the ability to cleave DNA from Cas9.

The new approach developed by researchers led by George Church PhD, of Harvard University and Ron Weiss PhD, of the Massachusetts Institute of Technology, allows both tasks to be achieved using one type of Cas9.

This new Cas9 allows scientists to increase the complexity of gene editing functions and increases overall control of targeted genes. It also opens up the possibility for better understanding how drug mechanisms affect diseases.

The findings are published in the September 7 issue of Nature Methods.

A multi-institutional team° introduced this new Cas9 protein. Its trick is to simultaneously cleave a gene while also regulating the expression of other genes — all done through reengineering the guide RNAs. Key to the  team's strategy was the discovery that the length of the guide RNA sequence is critical in whether Cas9 either binds to or simply cuts DNA.

"We decided to systematically test why it was that too much truncating of RNA guides caused Cas9 to no longer cut the intended gene site."

Alejandro Chavez, Postdoctoral Associate in Ron Weiss' MIT lab, the Wyss Institute, also advised by both George Church and James Collins at the Wyss, is a co-first author on the study together with Samira Kiani.

The Wyss/MIT team confirmed that in human cells, shorter guide RNAs do not allow Cas9 to cut a targeted gene. However, shorter guide RNAs also do not prevent Cas9 from binding to its gene target. Thus scientists can attach these gene regulation proteins to Cas9 which still binds them to specific genes.

"By using our newly uncovered guide RNA principle, we could for the first time, toggle a single protein to gain direct control over both gene sequences and gene expression — turning almost any DNA sequence into a regulatory sequence bent to our will.

"We envision future uses for the technology to help decipher the tangled web of interactions underlying, for example, cancer drug resistance and stem cell differentiation, or design advanced synthetic gene circuitries."

George Church PhD, Core Faculty member, Wyss Institute for Biologically Inspired Engineering, Harvard University.

"This new functionality will improve our ability to decipher the complex relationships between interdependent genes responsible for many diseases," said Marcelle Tuttle, a Research Fellow at the Wyss Institute and co-author on the study.

The findings could also be used in the large scale production of chemicals and fuels using genetically engineered bacteria — such as common E. coli — while safeguarding "microbial workers" from infection by other microbes and pathogens.

Abstract
We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

° Team Participants
George Church is a Core Faculty member at Harvard's Wyss Institute for Biologically Inspired Engineering, Robert Winthrop is Professor of Genetics at Harvard Medical School and Professor of Health Sciences and Technology at Harvard and MIT, and Ron Weiss is Professor of Biological Engineering and also Professor of Electrical Engineering and Computer Science at MIT. James Collins PhD, of the Wyss Institute Core Faculty member and the Termeer Professor of Medical Engineering & Science and Professor of Biological Engineering at MIT, is also a co-investigator and a co-author on the study.

The Wyss Institute for Biologically Inspired Engineering at Harvard University uses Nature's design principles to develop bioinspired materials and devices that will transform medicine and create a more sustainable world. Wyss researchers are developing innovative new engineering solutions for healthcare, energy, architecture, robotics, and manufacturing that are translated into commercial products and therapies through collaborations with clinical investigators, corporate alliances, and formation of new start-ups. The Wyss Institute creates transformative technological breakthroughs by engaging in high risk research, and crosses disciplinary and institutional barriers, working as an alliance that includes Harvard's Schools of Medicine, Engineering, Arts & Sciences and Design, and in partnership with Beth Israel Deaconess Medical Center, Brigham and Women's Hospital, Boston Children's Hospital, Dana-Farber Cancer Institute, Massachusetts General Hospital, the University of Massachusetts Medical School, Spaulding Rehabilitation Hospital, Boston University, Tufts University, and Charité - Universitätsmedizin Berlin, University of Zurich and Massachusetts Institute of Technology

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CRISPR-Cas9 is a new system using a naturally occuring protein, Cas9,
to cut DNA at specific sites and enable scientists to turn genes off or on.
Image Credit: Courtesy of the Broad Institute