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

Author

Listed:
  • Santiago Gisler

    (Oncode and The Netherlands Cancer Institute)

  • Joana P. Gonçalves

    (Delft University of Technology
    Oncode and The Netherlands Cancer Institute)

  • Waseem Akhtar

    (Oncode and The Netherlands Cancer Institute)

  • Johann de Jong

    (Oncode and The Netherlands Cancer Institute
    UCB Biosciences GmbH)

  • Alexey V. Pindyurin

    (Siberian Branch of Russian Academy of Sciences
    Oncode and The Netherlands Cancer Institute)

  • Lodewyk F. A. Wessels

    (Delft University of Technology
    Oncode and The Netherlands Cancer Institute)

  • Maarten van Lohuizen

    (Oncode and The Netherlands Cancer Institute)

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.

Suggested Citation

  • Santiago Gisler & Joana P. Gonçalves & Waseem Akhtar & Johann de Jong & Alexey V. Pindyurin & Lodewyk F. A. Wessels & Maarten van Lohuizen, 2019. "Multiplexed Cas9 targeting reveals genomic location effects and gRNA-based staggered breaks influencing mutation efficiency," Nature Communications, Nature, vol. 10(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-09551-w
    DOI: 10.1038/s41467-019-09551-w
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    Cited by:

    1. Jun Huang & David Rowe & Pratima Subedi & Wei Zhang & Tyler Suelter & Barbara Valent & David E. Cook, 2022. "CRISPR-Cas12a induced DNA double-strand breaks are repaired by multiple pathways with different mutation profiles in Magnaporthe oryzae," Nature Communications, Nature, vol. 13(1), pages 1-18, December.
    2. Trevor Weiss & Jitesh Kumar & Chuan Chen & Shengsong Guo & Oliver Schlegel & John Lutterman & Kun Ling & Feng Zhang, 2024. "Dual activities of an X-family DNA polymerase regulate CRISPR-induced insertional mutagenesis across species," Nature Communications, Nature, vol. 15(1), pages 1-12, December.

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