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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Multiplex Genome Engineering Using CRISPR/Cas Systems
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RNA-Guided Human Genome Engineering via Cas9
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DNA targeting specificity of RNA-guided Cas9 nucleases
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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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Improving CRISPR-Cas nuclease specificity using truncated guide RNAs
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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…”
BackgroundMethodInfluentialMali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., Yang, L., & Church, G. M. (2013) · Nature Biotechnology · doi:10.1038/nbt.2675 · Cited by 1,752 · cited 15×
“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).”
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“Mismatches in the PAM-distal nucleotides generally have less effect on disrupting the sgRNA-target DNA hybrid than those in the PAM-proximal nucleotides (PAMPNs) (Jinek et al., 2012; Cong et al., 2013; Fu et al., 2013; Mali et al., 2013; Pattanayak et al., 2013; Wu et al., 2014).”
BackgroundResultInfluentialStructures of Cas9 endonucleases reveal RNA-mediated conformational activation
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Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila
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Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells
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Heritable genome editing in C. elegans via a CRISPR-Cas9 system
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“Furthermore, we evaluated the mutation rates at potential off-target sites with two mismatches and one insertion/deletion to 13 sgRNAs (Lin et al., 2014), and again observed no off-target effects (Figure S1B).”
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Cited by
Haeussler, M., Schönig, K., Eckert, H., Eschstruth, A., Mianné, J., Renaud, J.-B., Schneider-Maunoury, S., Shkumatava, A., Teboul, L., Kent, J., et al. (2016) · Genome biology · doi:10.1186/s13059-016-1012-2 · Cited by 1,756
CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo
Moreno-Mateos, M. A., Vejnar, C. E., Beaudoin, J.-D., Fernandez, J. P., Mis, E. K., Khokha, M. K., & Giraldez, A. J. (2015) · Nature Methods · doi:10.1038/nmeth.3543 · Cited by 1,290
Off-target Effects in CRISPR/Cas9-mediated Genome Engineering
Zhang, X.-H., Tee, L. Y., Wang, X.-G., Huang, Q.-S., & Yang, S.-H. (2015) · Molecular Therapy - Nucleic Acids · doi:10.1038/mtna.2015.37 · Cited by 1,273
Self-citationCRISPR-P 2.0: An Improved CRISPR-Cas9 Tool for Genome Editing in Plants
Liu, H., Ding, Y., Zhou, Y., Jin, W., Xie, K., & Chen, L.-L. (2017) · Molecular Plant · doi:10.1016/j.molp.2017.01.003 · Cited by 837
Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing
Naeem, M., Majeed, S., Hoque, M. Z., & Ahmad, I. (2020) · Cells · doi:10.3390/cells9071608 · Cited by 457
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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