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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Vydáno v:	Nature communications Ročník 10; číslo 1; s. 1598 - 14
Hlavní autoři:	Gisler, Santiago, Gonçalves, Joana P., Akhtar, Waseem, de Jong, Johann, Pindyurin, Alexey V., Wessels, Lodewyk F. A., van Lohuizen, Maarten
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	London Nature Publishing Group UK 08.04.2019 Nature Publishing Group Nature Portfolio
Témata:	38/39 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
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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. 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.
AbstractList	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. 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. 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.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. 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. 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.
ArticleNumber	1598
Author	van Lohuizen, Maarten Akhtar, Waseem Gonçalves, Joana P. Pindyurin, Alexey V. Gisler, Santiago Wessels, Lodewyk F. A. de Jong, Johann
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BackLink	https://www.ncbi.nlm.nih.gov/pubmed/30962441$$D View this record in MEDLINE/PubMed
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Snippet	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... Designing effective genome engineering strategies requires an understanding of the impact that genomic locus has on CRISPR-Cas9 activity. Here the authors use...
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SubjectTerms	38/39 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
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Title	Multiplexed Cas9 targeting reveals genomic location effects and gRNA-based staggered breaks influencing mutation efficiency
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