Accurate characterization of CRISPR-Cas9 genome editing outcomes and mosaicism with near-perfect long reads

Background: Genetic mosaicism is a consequence of CRISPR-Cas9 genome editing that is difficult to study, especially when it involves structural variants occurring at low frequency. A comprehensive analysis of mosaicism requires deep and unbiased sequencing of the target loci, with accurate single molecule reads. Here we performed amplification-free PureTarget PacBio sequencing to investigate CRISPR-Cas9 outcomes at on-target and off-target sites in germline edited zebrafish and their offspring. Results: Thirty samples from pooled larvae and individual zebrafish were successfully sequenced, resulting in >1100x average target coverage. The PacBio reads reached an exceptional accuracy (QV39) over the target regions, with every read originating from a unique DNA molecule. The two haplotypes of the target loci displayed a balanced depth of coverage, while long-range PCR of the same samples resulted in skewed data. Further analysis of the PureTarget data revealed widespread genetic mosaicism in individual founder (F0) fish, with up to 18 distinct on-target events and 11 off-target events present in a single adult founder. Several CRISPR-Cas9 editing outcomes, including large structural variants and off-target mutations, were inherited to the F1 generation. Notably, as many as seven unique editing events were found among F1 juvenile offspring from a single founder pair, thereby confirming the presence of genetic mosaicism in germ cells of founder zebrafish. This implies that some consequences of CRISPR-Cas9 editing may emerge only in the second generation. We also analyzed DNA methylation but did not observe altered 5mC CpG signals in genome edited samples. Conclusions: PureTarget enables efficient, accurate and unbiased analysis of the full spectrum of genetic mosaicism in a sample. The potential risks of introducing germ cell mosaicism in founder individuals should be carefully evaluated when designing germline genome editing experiments.