In vitro CRISPR system allows scientists to detect broad outcomes

By Samantha Black, PhD, The Science Advisory Board editor in chief

As CRISPR technology advances, it is crucial to identify all unintended and potentially harmful changes introduced by gene-editing processes. A new study published in Communications Biology introduces a new system that details broad spectrum outcomes of gene-editing reactions in an unbiased fashion.

Clustered regularly interspersed palindromic repeats (CRISPR) and CRISPR-associated Cas nucleases have provided researchers with novel tools to correct single-base mutations or gene deletions that can be life-changing. Specifically, when DNA is cleaved, several error-prone reactions, such as non-homologous end joining (NHEJ) and homology-directed repair (HDR) compete at the site. Clinicians need to know all outcomes of specific gene-editing reactions to enable more educated choices of the type and amount of genetic engineering to employ for treatment of a genetic disorder.

"We've developed a new process for rapidly screening all of the edits made by CRISPR, and it shows there may be many more unintended changes to DNA around the site of a CRISPR repair than previously thought," said Eric Kmiec, PhD, director of ChristianaCare's Gene Editing Institute and the principle author of the study.

The researchers used a system that employs mammalian cell-free extract to drive gene-editing reactions and control the level of reaction in order to catalog the distribution of insertions, deletions, and duplications as the function of a specific genetic tool. Using plasmids, the scientists were able to eliminate much of the biological complexity that makes it difficult to isolate the full array of DNA changes introduced by CRISPR. The tool can provide results in just 48 hours, expediting a process that often requires up to two months of expensive and complicated DNA analysis. The tool also allows scientists to execute and screen multiple trial edits and catch unintended mutations that they would not otherwise notice.

"It's important to note that in all instances we were still seeing CRISPR achieve a fantastic level of successful repairs that would have been unimaginable even five years ago," added lead author Brett Sansbury. "But we saw a lot of other changes to DNA near the site of the repair that needs to be better understood so that when we correct one problem, we're not creating another." They believe that the vast majority of unintended edits may have no consequence for patients, but it's important to identify them and determine which ones might pose a risk.

The researchers observed error-prone events in multiple CRISPR templates, including Cas9, Cas12a, 1364-S, and 1364-NS, all of which had unique reaction profiles. The rate of "precise repair"--repairs that are accomplished without introducing unintended mutations--varied considerably depending on the enzyme and template employed, ranging from a low of five percent to a high of 64 percent.

"CRISPR will probably never be perfect 100 percent of the time," Kmiec said. "But CRISPR tools are constantly improving. And if we can achieve a 70 or 80 percent rate of precision--and reveal and understand the importance of any changes that occur alongside that repair--that brings us much closer to safely using CRISPR to treat patients. We hope our new tool can help accelerate efforts to achieve that goal."

The researcher's greater goal is to develop better CRISPR screening systems in DNA plasmids extracted from human cells. They have already used this cell-free approach to engineer multiple edits simultaneous and have partnered with a biotech firm to developed personalized cancer treatments.

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