Discovery and characterization of sgRNA-independent DNA cleavage from CRISPR/Cas9 in mouse embryos

Background: The CRISPR/Cas9 system can induce off-target effects in programmed gene editing, but there have been few reports on cleavage detection and characterization in early embryos. To study these events, sgRNAs with different off-target rates were designed by off-target prediction software and injected into mouse zygotes with Cas9. The development of the injected embryos was analyzed, and γH2AX, which can bind sequences at double-strand breaks (DSBs), was used for DNA cleavage analysis by immunostaining and CUT&Tag. Results The results showed that the sgRNAs with a higher off-target rate were associated with significantly reduced blastocyst rate and birth rate in injected mouse embryos. γH2AX immunofluorescence indicated that there was a relative DSB peak at 15 h after Cas9 system injection, and the number of γH2AX foci at the peak was significantly higher in the low off-target sgRNA-injected group (101.3 ± 16.6) than in the control group (42.2 ± 9.9). No predicted off-target mutations with a difference of 4 bases from the sgRNA were found in the Cas9-edited mice or fetuses. At 15 h after CRISPR/Cas9 and low off-target sgRNA injection, many sgRNA-independent DSBs were detected by CUT&Tag sequencing. The distribution of sgRNA-independent DSBs had no chromosome specificity. Gene Ontology (GO) annotation analysis of the sgRNA-independent DSB sites showed that the sgRNA-independent DSBs were mainly concentrated at genes associated with biological processes such as neuron projection morphogenesis and steroid hormone-mediated signaling; molecular functions such as PDZ domain binding and actin binding; and cell components such as postsynaptic membrane and intracellular vessel. Conclusion In conclusion, the many sgRNA-independent cleavages induced when the Cas9 system is used for gene editing in mouse embryos were visually detected and characterized in the genome for the first time. These results should also be considered when using or optimizing the Cas9 system.