A concern that has plagued CRISPR-Cas9 technology from the beginning is the possibility, and unknown effect, of off-target effects—DNA mutations that result from CRISPR-Cas9 editing that are not in the targeted area. In order to understand the effect of off-target changes, they must first be detected. A new universally applicable approach for unbiased off-target identification has been developed. Named DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), it leverages the recruitment of DNA repair factors in cells and organisms.

The work, from groups at the Innovative Genomics Institute (IGI) at the University of California, Berkeley, the Gladstone Institutes in San Francisco, and AstraZeneca in Sweden, is reported Science in the paper titled, “Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq.”

“When CRISPR makes a cut, the DNA is broken,” said Beeke Wienert, PhD, a postdoctoral researcher at IGI and first author on the paper. “So, in order to survive, the cell recruits many different DNA repair factors to that particular site in the genome to fix the break and join the cut ends back together. We thought that if we could find the locations of these DNA repair factors, we could identify the sites that have been cut by CRISPR.”

The researchers investigated different DNA repair proteins for their ability to identify Streptococcus pyogenes Cas9 target sites by ChIP-Seq. They focused on the MRE11 subunit of the MRN complex, one of the first repair protein recruited to the site of the cut and is found to be “tightly distributed around the Cas9 cut site.” The authors write that “MRE11 binding peaked before the appearance of insertions and deletions (indels) and was readily detected at a known guide RNA (gRNA) off-target.” Using MRE11, DISCOVER-Seq can identify the exact sites in the genome where a cut has been made by CRISPR. Most MRE11 ChIP-Seq reads precisely ended at the predicted Cas9 cut, enabling identification of the nuclease site with single-base resolution. The authors write that, “tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution.”

“The human genome is extremely large—if you printed the entire DNA sequence, you would end up with a novel as tall as a 16-story building,” explained Bruce Conklin, MD, senior investigator at Gladstone and deputy director at IGI. “When we want to cut DNA with CRISPR, it’s like we’re trying to remove one specific word on a particular page in that novel.”

“You can think of the DNA repair factors as different types of bookmarks added to the book,” Conklin added. “While some may bookmark an entire chapter, MRE11 is a bookmark that drills down to the exact letter that has been changed.”

Different methods currently exist to detect CRISPR off-target effects. However, they come with limitations that range from producing false positive results to killing the cells they’re examining. In addition, the most common method used to date is currently limited to being used in cultured cells in the laboratory, excluding its use in patient-derived stem cells or animal tissue.

“Because our method relies on the cell’s natural repair process to identify cuts, it has proven to be much less invasive and much more reliable,” said Jacob Corn, PhD, who now runs a laboratory at ETH Zurich. “We were able to test our new DISCOVER-Seq method in induced pluripotent stem cells, patient cells, and mice, and our findings indicate that this method could potentially be used in any system, rather than just in the lab.”

DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing. By being applied to new cell types and systems, DISCOVER-Seq has also revealed new insights into the mechanisms used by CRISPR to edit the genome, which will lead to a better understanding of the biology of how this tool works.

“The new method greatly simplifies the process of identifying off-target effects while also increasing the accuracy of the results,” said Conklin. “This could allow us to better predict how genome editing would work in a clinical setting. As a result, it represents an essential step in improving preclinical studies and bringing CRISPR-based therapies closer to the patients in need.”

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