Cas9-induced on-target genomic damage

CRISPR/Cas9 is the gene editing tool of choice in basic research and poised to become one in clinical context. However, current studies on the topic suffer from a number of shortcomings. Mutagenesis is often assessed using bulk methods, which means rare events go undetected, unresolved or are discarded as potential sequencing errors. Many of the genotyping methods rely on short-range PCR, which excludes larger structural variants. Other methods, such as FISH, do not provide basepair resolution, making the genotype assessment imprecise. Furthermore, it is not well understood how Cas9 delivery format influences the dynamics of indel introduction. Finally, many studies of on-target activity were conducted in cancerous cell lines, which do not accurately model the mutagenesis of normal cells in the therapeutic context. In my thesis, I have investigated on-target lesions induced by Cas9 complexed with single gRNAs and no exogenous template. I have followed the time dynamics of Cas9-induced small indels as a function of reagent delivery methods, established an assay for quantification of Cas9-induced genomic lesions that are not small indels ("complex lesions") and used this assay to isolate and genotype complex lesions, many of which would be missed by standard methods. I found that DNA breaks introduced by single guide RNAs frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and cross-over events were identified. Frequent and extensive DNA damage in mitotically active cells caused by CRISPR/Cas9 editing may have pathogenic consequences.