Tsai, Shengdar Q.Nguyen, NhuMalagon-Lopez, JoseTopkar, Ved V.Aryee, MartinJoung, J. Keith2018-05-302017Tsai, Shengdar Q., Nhu T. Nguyen, Jose Malagon-Lopez, Ved V. Topkar, Martin J. Aryee, and J. Keith Joung. 2017. “CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets.” Nature methods 14 (6): 607-614. doi:10.1038/nmeth.4278. http://dx.doi.org/10.1038/nmeth.4278.http://nrs.harvard.edu/urn-3:HUL.InstRepos:37068196Sensitive detection of off-target effects is important for translating CRISPR-Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe CIRCLE-seq (Circularization for In vitro Reporting of CLeavage Effects by sequencing), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR-Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequence. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific SNPs, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides the most accessible, rapid and comprehensive method for identifying genome-wide off-target mutations of CRISPR-Cas9 described to date.en-USJournal Article2018-05-3010.1038/nmeth.4278