Print Email Facebook Twitter Multiplexed Cas9 targeting reveals genomic location effects and gRNA-based staggered breaks influencing mutation efficiency Title Multiplexed Cas9 targeting reveals genomic location effects and gRNA-based staggered breaks influencing mutation efficiency Author Gisler, Santiago (Netherlands Cancer Institute) P. Gonçalves, Joana (TU Delft Pattern Recognition and Bioinformatics; Netherlands Cancer Institute) Akhtar, Waseem (Netherlands Cancer Institute) de Jong, Johann (Netherlands Cancer Institute; UCB Biosciences GmbH) Pindyurin, Alexey V. (Russian Academy of Sciences; Netherlands Cancer Institute) Wessels, L.F.A. (TU Delft Pattern Recognition and Bioinformatics; Netherlands Cancer Institute) van Lohuizen, Maarten (Netherlands Cancer Institute) Date 2019 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. To reference this document use: http://resolver.tudelft.nl/uuid:dd034c4d-07bf-4b29-b382-33cdae30e0f6 DOI https://doi.org/10.1038/s41467-019-09551-w ISSN 2041-1723 Source Nature Communications, 10 (1), 1-14 Part of collection Institutional Repository Document type journal article Rights © 2019 Santiago Gisler, Joana P. Gonçalves, Waseem Akhtar, Johann de Jong, Alexey V. Pindyurin, L.F.A. Wessels, Maarten van Lohuizen Files PDF 53249812_s41467_019_09551_w.pdf 2.19 MB Close viewer /islandora/object/uuid:dd034c4d-07bf-4b29-b382-33cdae30e0f6/datastream/OBJ/view