sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing
Summary: Cas9 nucleases can be programmed with single guide RNAs (sgRNAs) to mediate gene editing. High CRISPR/Cas9-mediated gene knockout efficiencies are essential for genetic screens and critically depend on the properties of the sgRNAs used. The specificity of an sgRNA is defined by its targetin...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2019-01-01
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| Series: | Cell Reports |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2211124719300336 |
| _version_ | 1818450410633953280 |
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| author | Robin Graf Xun Li Van Trung Chu Klaus Rajewsky |
| author_facet | Robin Graf Xun Li Van Trung Chu Klaus Rajewsky |
| author_sort | Robin Graf |
| collection | DOAJ |
| description | Summary: Cas9 nucleases can be programmed with single guide RNAs (sgRNAs) to mediate gene editing. High CRISPR/Cas9-mediated gene knockout efficiencies are essential for genetic screens and critically depend on the properties of the sgRNAs used. The specificity of an sgRNA is defined by its targeting sequence. Here, we discovered that two short sequence motifs at the 3′ end of the targeting sequence are almost exclusively present in inefficient sgRNAs of published sgRNA-activity datasets. By specific knock-in of sgRNA target sequences with or without these motifs and quantitative measurement of knockout efficiency, we show that the presence of these motifs in sgRNAs per se results in a 10-fold reduction of gene knockout frequencies. Mechanistically, the cause of the low efficiency differs between the two motifs. These sequence motifs are relevant for future sgRNA design approaches and studies of Cas9-DNA interactions. : CRISPR/Cas9-mediated gene editing efficiency critically depends on the properties of the sgRNAs used. Graf et al. show that the presence of two position-specific sequence motifs in the targeting sequence of sgRNAs per se results in a severe reduction of gene knockout frequencies. Keywords: CRISPR/Cas9, gene targeting, knockout efficiency, sgRNA design, sgRNA efficiency, sgRNA motif, scaffold RNA, CrispRGold, CRISPR screening |
| first_indexed | 2024-12-14T20:50:52Z |
| format | Article |
| id | doaj.art-97f82ac63e3c4966940b138cc9cd8188 |
| institution | Directory Open Access Journal |
| issn | 2211-1247 |
| language | English |
| last_indexed | 2024-12-14T20:50:52Z |
| publishDate | 2019-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Reports |
| spelling | doaj.art-97f82ac63e3c4966940b138cc9cd81882022-12-21T22:47:48ZengElsevierCell Reports2211-12472019-01-0126510981103.e3sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene EditingRobin Graf0Xun Li1Van Trung Chu2Klaus Rajewsky3Max Delbrück Center for Molecular Medicine, Berlin, Germany; Corresponding authorMax Delbrück Center for Molecular Medicine, Berlin, GermanyMax Delbrück Center for Molecular Medicine, Berlin, Germany; Berlin Institute of Health, Berlin, GermanyMax Delbrück Center for Molecular Medicine, Berlin, GermanySummary: Cas9 nucleases can be programmed with single guide RNAs (sgRNAs) to mediate gene editing. High CRISPR/Cas9-mediated gene knockout efficiencies are essential for genetic screens and critically depend on the properties of the sgRNAs used. The specificity of an sgRNA is defined by its targeting sequence. Here, we discovered that two short sequence motifs at the 3′ end of the targeting sequence are almost exclusively present in inefficient sgRNAs of published sgRNA-activity datasets. By specific knock-in of sgRNA target sequences with or without these motifs and quantitative measurement of knockout efficiency, we show that the presence of these motifs in sgRNAs per se results in a 10-fold reduction of gene knockout frequencies. Mechanistically, the cause of the low efficiency differs between the two motifs. These sequence motifs are relevant for future sgRNA design approaches and studies of Cas9-DNA interactions. : CRISPR/Cas9-mediated gene editing efficiency critically depends on the properties of the sgRNAs used. Graf et al. show that the presence of two position-specific sequence motifs in the targeting sequence of sgRNAs per se results in a severe reduction of gene knockout frequencies. Keywords: CRISPR/Cas9, gene targeting, knockout efficiency, sgRNA design, sgRNA efficiency, sgRNA motif, scaffold RNA, CrispRGold, CRISPR screeninghttp://www.sciencedirect.com/science/article/pii/S2211124719300336 |
| spellingShingle | Robin Graf Xun Li Van Trung Chu Klaus Rajewsky sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing Cell Reports |
| title | sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing |
| title_full | sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing |
| title_fullStr | sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing |
| title_full_unstemmed | sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing |
| title_short | sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing |
| title_sort | sgrna sequence motifs blocking efficient crispr cas9 mediated gene editing |
| url | http://www.sciencedirect.com/science/article/pii/S2211124719300336 |
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