Genuine-seq enables genome-wide unbiased identification of off-target cleavage and translocation by CRISPR nucleases, II

In practice, CRISPR’s causes chromosomal structural variation due to off-targets, which is a serious genotoxicity, while currently unbiased methods for detecting chromosomal structural variation are largely lacking. Here, we developed Genome-wide ubiased identification of nuclease effects by sequencing (Genuine-Seq), which could detect both off-target sites of CRISPR and chromosomal structural variations caused by off-target effects, including translocations and large deletions.

We demonstrated Genuine-seq’s high efficiency in human cells, and compared spCas9 and spRY, emphasizing that spRY can cause tremendous off-target cutting. Using mouse models, we performed in vivo experiments to depict the landscape of off-target sites and chromosomal structural variations caused by CRISPR-Cas editing, which demonstrated the translational value of Genuine-seq in safety assessment of CRISPR-based therapeutics.