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

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 repor...

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Bibliographic Details
Published in:Nature communications Vol. 10; no. 1; pp. 1598 - 14
Main Authors: Gisler, Santiago, Gonçalves, Joana P., Akhtar, Waseem, de Jong, Johann, Pindyurin, Alexey V., Wessels, Lodewyk F. A., van Lohuizen, Maarten
Format: Journal Article
Language:English
Published: London Nature Publishing Group UK 08.04.2019
Nature Publishing Group
Nature Portfolio
Subjects:
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38/47
38/70
45/41
631/1647/1513/1967/3196
631/1647/2017/1958
631/337/176
631/337/4041
Animals
Cell Line
CRISPR
CRISPR-Cas Systems - genetics
Deoxyribonucleic acid
DNA
DNA - genetics
DNA Mutational Analysis
DNA repair
Efficiency
Embryo cells
Gene Editing - methods
Gene sequencing
Genes, Reporter - genetics
Genetic Loci - genetics
Genetic modification
Genetic Vectors - genetics
Genome editing
Genomes
Genomics
gRNA
Humanities and Social Sciences
Loci
Lymphocytes B
Mice
Mouse Embryonic Stem Cells
multidisciplinary
Mutation
Mutation Rate
Nucleotide sequence
Plasmids - genetics
Ribonucleic acid
RNA
RNA, Guide, CRISPR-Cas Systems - genetics
Science
Science (multidisciplinary)
Stem cell transplantation
Stem cells
ISSN:2041-1723, 2041-1723
Online Access:Get full text
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Summary: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. Designing effective genome engineering strategies requires an understanding of the impact that genomic locus has on CRISPR-Cas9 activity. Here the authors use TRIP integrations to profile editing outcomes genome-wide and observe that gRNA sequence influences the structure of the double strand break.
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ISSN:2041-1723
2041-1723
DOI:10.1038/s41467-019-09551-w