Chromosomal Engineering By Chemical Induced Proximity and Cas9-Mediated DNA Recombination

Chromosomal translocations affect a number of human disorders, including many cancers such as Burkitt’s lymphoma and synovial sarcoma, as well as developmental disorders such as autism and intellectual disability. Most translocations are thought to involve inaccurate DNA repair following a double-stranded break; however, the mechanisms of acquired translocations and their resulting pathology are unclear. Despite identifying over 10,000 chromosomal fusions in human cancers and other genetic diseases, we currently lack faithful models to study such diseases. In this work, we integrate CRISPR/Cas9 with a chemically-induced dimerization (CID) system to generate desired chromosomal rearrangements resulting in endogenous expression of fusion proteins. Chemically-induced dimerization (CID), a biochemical strategy to regulate molecular proximity within the cell, uses bifunctional small molecules to dimerize two proteins of interest. Here, we describe a small-molecule chemical homodimerizer, FK1012, that rapidly co-localizes two Cas9 proteins targeted to distinct genomic regions to generate a balanced translocation. As an example, we show that time-dependent translocations induce RNA expression of the Npm1-Alk fusion protein found in 30-50% of anaplastic large-cell non-Hodgkin’s lymphomas. Ultimately, our technology could yield targeted rearrangements for therapeutic investigations and provide a method to dissect the mechanisms of genomic translocations.