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

Santiago Gisler, Joana P. Gonçalves, Waseem Akhtar, Johann de Jong, Alexey V. Pindyurin, Lodewyk F. A. Wessels () and Maarten van Lohuizen ()
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Santiago Gisler: Oncode and The Netherlands Cancer Institute
Joana P. Gonçalves: Delft University of Technology
Waseem Akhtar: Oncode and The Netherlands Cancer Institute
Johann de Jong: Oncode and The Netherlands Cancer Institute
Alexey V. Pindyurin: Siberian Branch of Russian Academy of Sciences
Lodewyk F. A. Wessels: Delft University of Technology
Maarten van Lohuizen: Oncode and The Netherlands Cancer Institute

Nature Communications, 2019, vol. 10, issue 1, 1-14

Abstract: Abstract 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. Here 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.

Date: 2019
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DOI: 10.1038/s41467-019-09551-w

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