The DOI matches its Crossref record; 19 of the 19 references it cites were found in a registry or an index.
Enhanced Specificity and Efficiency of the CRISPR/Cas9 System with Optimized sgRNA Parameters in Drosophila
Open accessAbstract
The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, single-guide RNA (sgRNA) parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer-adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair and achieved an overall mutagenesis rate significantly higher than previously reported. Our work demonstrates a comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.
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Cites
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High-Frequency off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells
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“The off-target effect (i.e., specificity) and efficiency of the CRISPR/Cas9 system has been recently investigated in mammalian cells (Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013; Pattanayak et al., 2013; Cho et al., 2014; Fu et al., 2014; Kuscu et al., 2014; Lin et al., 2014; Wu et al.,…”
BackgroundInfluentialRNA-guided editing of bacterial genomes using CRISPR-Cas systems
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Crystal structure of Cas9 in complex with guide RNA and target DNA
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“…nt A previous report shows that removing the two nucleotides most distal to the PAM from the sgRNA does not interfere with efficiency significantly (Fu et al., 2014), but mutagenesis efficiency at genomic sites with two contiguous mismatches in these nucleotides is compromised in our assays…”
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“This conclusion is further supported by the observation that sgRNAs targeting the y, w, v, and e genes do not induce off-target mutations in genomic DNA carrying three mismatches (Figure 1D and Figure S1B), and is in line with recent studies in mammalian cells (Hsu et al., 2013; Mali et al., 2013; Wang et al., 2014).”
BackgroundResultInfluential
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Okada, K., Aoki, K., Tabei, T., Sugio, K., Imai, K., Bonkohara, Y., & Kamachi, Y. (2022) · Nucleic Acids Research · doi:10.1093/nar/gkac100 · Cited by 14
(2016) · doi:10.13918/j.issn.2095-8137.2016.4.191
Metadata
- Authors
- Ren, X., Yang, Z., Xu, J., Sun, J., Mao, D., Hu, Y., Yang, S.-J., Qiao, H.-H., Wang, X., Hu, Q., Deng, P., Liu, L.-P., Ji, J.-Y., Li, J. B., & Ni, J.-Q.
- Venue
- Cell Reports
- Publisher
- Elsevier BV
- Published
- 2014-11
- Volume
- 9
- Issue
- 3
- Pages
- 1151-1162
- ISSN
- 2211-1247 (ISSN-L)
- OpenAlex
- W1971305813
- Cited by
- 309
- References
- 19
- FWCI
- 11.44
- Type
- journal article
- Language
- en
- Open access
- gold
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