Multiplexed Cas9 targeting reveals genomic location effects and gRNA-based staggered breaks influencing mutation efficiency and patterns

Understanding the impact of guide RNA (gRNA) and genomic locus on CRISPR/Cas9 activity is crucial to design effective gene editing assays. However, it is challenging to profile Cas9 activity in the endogenous cellular environment. We leverage our TRIP technology to integrate ~1k barcoded reporter genes in the genomes of mouse embryonic stem cells. We target the integrated reporters (IRs) using RNA-guided Cas9 and characterize induced mutations by sequencing. We report that gRNA-sequence and IR locus explain most variation in mutation efficiency. Predominant insertions of a gRNA-specific nucleotide are consistent with template-dependent repair of staggered DNA ends with 1-bp 5' overhangs. We confirm that such staggered ends are induced by Cas9 in mouse pre-B cells. To explain observed insertions, we propose a model generating primarily blunt and occasionally staggered DNA ends. Mutation patterns indicate that gRNA-sequence controls the fraction of staggered ends, which could be used to optimize Cas9-based insertion efficiency. Overall design: Mutation profiling upon RNA-guided Cas9 targeting of (i) mouse embryonic stem (mES) cell line, EStTA-PB-B18, containing the same 36 mPGK-driven TRIP barcoded reporter integrations per cell (EStTA-PB-B18); (ii) TRIP cell pool containing ~1k barcoded reporters integrated heterogeneously throughout the genomes of the cells in the pool (GEMP-O). Cell line was targeted in independent experiments using three single-guide RNAs (sgRNA1-3). Pool was targeted in independent experiments using sgRNA2 with or without a custom-designed ssODN, and with sgRNA3. We also did expression profiling of the TRIP cell line and pool before Cas9 targeting (see GEO accession GSE48606 for cell line expression, and this submission for the pool expression). Files related to re-analyzed samples from GSE48606 are linked below as supplementary files.