A microfluidic platform for the characterization of CRISPR “off-target” activity

engineering of any genomic DNA sequence. The engineered nucleases, such as CRISPR- Cas9, generate genomic site-specific double-stranded breaks (DSBs), which then can be resolved by the cell’s endogenous DNA repair mechanisms. This is mediated by a 20 nucleotide hybridization of a guide RNA to a target DNA sequence. Cas9-mediated DNA cleavage specificity, both in vitro and in vivo, has been inferred previously based on assays against small collections of potential single-mutation off-target sites. These studies suggested that perfect complementarity between guide RNA and target DNA is required in the base pairs adjacent to the PAM end of the target site (3′ end of the guide RNA) while mismatches are tolerated at the non-PAM end (5′ end of the guide RNA). These mismatches create “off-target” DNA editing events. Such “off-target” events are a serious safety issue for any genetic therapy and thus are important to characterize. Additionally, “on target” efficiency varies between different guide RNAs, which makes it as important to characterize.

The present study, conducted in collaboration with Dr. Ayal Hendel`s lab, aim to create a microfluidics based assay to characterize the biochemistry of Cas9- gRNA complex binding and cleavage of the “on-target” and “off-target” DNA. Our integrated microfluidics technology, enables simultaneously performance of large scale complex assays, quickly and efficiently, with minimal co-contamination.

The following poster, will demonstrate initial proof-of-concept results of binding and cleavage assays performed on the microfluidic platform.