Abstract
CRISPR-Cas9 technology has been plagued by the possibility of off-target effects. Now an international team of researchers has developed a new universally applicable approach for unbiased off-target identification. Named DISCOVER-Seq, it leverages the recruitment of DNA repair factors in cells and organisms.
“When CRISPR makes a cut, the DNA is broken,” said Beeke Wienert, Ph.D., a postdoctoral researcher at the Innovative Genomics Institute and first author on the paper “Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq” published in Science. “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.”
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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.