Expanding the CRISPR Toolbox for New Healthcare Applications

crispr illustrationCRISPR/Cas9 was first successfully demonstrated as a tool for genome editing in human cells in 2013.  Since then, several advances have enabled the use of this technology to shut down a faulty gene, change a single DNA letter in a gene sequence, or add a new gene to an existing genome sequence. This summer has seen two important new additions to the growing CRISPR toolbox for manipulating genes and their expression.


One new improvement, demonstrated by researchers at ETH Zurich, enables the simultaneous editing of dozens — or potentially even hundreds — of genes. The researchers were able to edit 25 genes at once with the use of a stabilized plasmid structure capable of holding and processing multiple RNA molecules to target CRISPR enzymes to the desired gene locations. The new capability should allow researchers to increase the activity of certain genes while decreasing that of others, thus enabling the investigation and coordination of genetic programs underlying complex cell processes.


A second — potentially even more useful — breakthrough allows CRISPR to be employed for the first time to directly edit RNA. Targeting disease-linked mutations in RNA, which is relatively short-lived in the cell, could avoid many of the risks of gene editing (such as off-target editing and potential deleterious mutations) as well as ethical questions posed by making permanent changes to the genome. Turning on or off certain genes for limited periods would be especially useful for creating short-term CRISPR-based therapies aimed at spurring wound healing, or treating inflammation, pain, stroke and infectious diseases.


The new advance was demonstrated by CRISPR pioneer Feng Zhang and his laboratory at the Broad Institute. Using the Cas13 enzyme, Zhang’s team first used an approach called REPAIR (RNA editing for programmable Adenine to Inosine) to convert APOE4, a gene associated with a high risk of Alzheimer’s disease, to the benign APOE2. They then took the REPAIR-ed gene and evolved it in the laboratory to change cytosine to uridine, a process they called RESCUE (RNA editing for specific C to U exchange). They also further optimized RESCUE to reduce off-target editing while minimally disrupting on-target effects.


The RESCUE technology expands the use of CRISPR to targeting RNA-mediated protein activity and function, which are typically regulated post-gene translation. The research team showed that they could achieve a reversible change in specific cellular processes by using CRISPR RNA-editing to orchestrate a transitory spike in beta-catenin activation and cell growth. Such a temporary effect could potentially be used to promote wound healing while eliminating the threat of cancer associated with uncontrolled cell growth.