Illuminating Cas9 scission profile for precise genome editing
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Abstract
CRISPR/Cas9 is a powerful genome editing platform, holding immense potential for successful gene therapy of various genetic diseases. Cas9 can generate different types of DNA double-strand breaks (DSBs) – blunt and staggered; however, what dictates Cas9 scission decision is largely unknown. Furthermore, it has been reported that the DSB end structure of the Cas9-induced cut has a direct effect on the repair outcome of loci targeted for gene editing. The scission profile remains an overlooked feature of Cas nucleases, and frequencies and determinants of the DSB end structures remain elusive.
Here we developed BreakTag, a versatile, highly parallel and scission-aware methodology for the profiling of Cas9-induced DSBs to identify molecular determinants influencing Cas9 incisions. Beyond testing the fidelity of gRNAs and nucleases, BreakTag is a straightforward framework for the characterization of CRISPR-Cas nucleases. Assessing the end-structure of more than 150,000 uniquely cleaved by Cas9 loci across the human genome, we found that in addition to frequent blunt DSBs, approximately 34% of SpCas9 cut sites form staggered ends displaying 1 to 3 nucleotide 5’ overhangs. The presence of mismatches between the gRNA and the target DNA influenced the scission profile, and the ratios of blunt and staggered cuts were target-dependent. Training a machine learning model that predicts Cas9 scission profile revealed that the nuclease incision is highly dependent on the protospacer sequence, with strong sequence determinants. We further demonstrated that genetic variation impacts Cas9 cut configuration and therefore, the DNA repair outcome. Using BreakTag, we identified high-fidelity Cas9 variants with altered scission profile properties, expanding the loci amenable for highly staggered cleavage. Comparing matched datasets of Cas9 incisions and repair outcome, we established that Cas9 staggered breaks are linked with precise, templated and predictable single-nucleotide insertions, indicating that controlling Cas9 staggered cut profile could allow prediction of repair genotypes with desirable indels. We demonstrated in a proof-of-principle experiment that a scission-aware gRNA design can be leveraged for correcting pathogenic single-nucleotide deletions, demonstrating the clinical application of staggered cleavage.
Our work illuminates fundamental characteristics of the Cas9 nuclease and lays the foundation for harnessing the flexible Cas9 cut profile and engineered variants for precise template-free genome editing.