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Policing the editor: Stanford scientists devise way to monitor CRISPR effectiveness

edited paperAs a writer, I think a lot about editing. Will this sentence work here? Maybe I should change this word. Argh - a typo! But I'm not alone. Biologists also appreciate the power of editing, particularly when it comes to modifying genes in cells or organisms.

Recently a powerful new technology has emerged (called CRISPR) that allows researchers to make small, precise and permanent changes in the DNA of animal and human cells. It builds on the concept of genome editing that is key to generating cells, cell lines or even whole animals such as laboratory mice, containing specific genetic changes for study. With CRISPR, however, researchers can generate in days or weeks experimental models that usually take months or years. As a result, they can quickly assess the effect of a particular gene by deleting it entirely, or experiment with repeated, tiny changes to its DNA sequence.

According to a recent New York Times article, scientists roundly agree that CRISPR is revolutionary. At least three companies have been launched in the mere 18 months since the first results were reported by researchers at the University of California, Berkeley and Umea University in Sweden, and more than 100 research papers based on the technique have been published. But, although it's highly specific, it's (sadly) not perfect. According to the New York Times piece:

Quick is not always accurate, however. While Crispr is generally precise, it can have off-target effects, cutting DNA at places where the sequence is similar but not identical to that of the guide RNA.

Obviously it's important to know when (and how frequently) this happens. Unfortunately, that's been difficult to assess.

Enter researchers in the laboratory of pediatric cancer biologist Matthew Porteus, MD, PhD. Porteus's lab is interested in (among other things) learning how to a particular type of genome editing called homologous recombination to treat diseases like sickle cell anemia, thalassemia, hemophilia and HIV. They've devised a way to monitor the efficiency of genome editing by CRISPR (as well as other more-traditional genome editing technologies) that could be widely helpful to researchers worldwide. Their technique was published today in Cell Reports. As postdoctoral researcher Ayal Hendel, PhD, told me:

We have developed a novel method for quantifying individual genome editing outcomes at any site of interest using single-molecule real-time (also known as SMRT) DNA sequencing. This approach works regardless of the editing technique used, and in any type of cell from any species.

Hendel teamed up with Eric Kildebeck, PhD, an MD/PhD student formerly in the Porteus lab, and Eli Fine, a graduate student at the Georgia Institute of Technology and Emory University, to conduct the work. Their approach is based on the ability of SMRT to sequence the junction points of the edited region in individual molecules. Researchers can compare the results from sequencing a population of cells to estimate the prevalence of the desired change (versus others in which the editing went awry) to judge the effectiveness of CRISPR or other genome editing - a capability that is sorely needed in the stampede to bring the power of CRISPR to human therapies.

The authors conclude in their paper:

The recent explosion in custom gene-editing technologies is ushering in a new age of genome engineering where scientists across fields of study and using different organisms and cell types can precisely modify essentially any locus they desire. Here, we show that SMRT DNA sequencing provides a simple, rapid, quantitative, and sensitive strategy for measuring genome-editing outcomes with different cell lines, at any endogenous loci, including transcriptionally silent loci, and using multiple nuclease platforms. [...] SMRT DNA sequencing can streamline the development of genome-editing projects and hasten the expansion of these technologies to a wider range of applications.

Previously: Gene "editing" could correct a host of genetic disorders and Engineering immune cells to resist HIV

Photo by Nic McPhee

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