The DOI matches its Crossref record; 14 of the 14 references it cites were found in a registry or an index.
High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9
Open accessAbstract
The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published “rules” for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5′ end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.
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Cites
The Sequence Alignment/Map format and SAMtools
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Cutadapt removes adapter sequences from high-throughput sequencing reads
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A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116
Multiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339
ggplot2: Elegant Graphics for Data Analysis
Wickham, H. (2009) · doi:10.1007/978-0-387-98141-3 · Cited by 14,447
RNA-Guided Human Genome Engineering via Cas9
Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., & Church, G. M. (2013) · Science · doi:10.1126/science.1232033 · Cited by 9,210
The zebrafish reference genome sequence and its relationship to the human genome.
Howe, K., Clark, M. D., Torroja, C. F., Torrance, J., Berthelot, C., Muffato, M., Collins, J. E., Humphray, S., McLaren, K., Matthews, L., et al. (2013) · Nature · doi:10.1038/nature12111 · Cited by 5,100
Self-citationDNA targeting specificity of RNA-guided Cas9 nucleases
Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y., Fine, E. J., Wu, X., Shalem, O., et al. (2013) · Nature Biotechnology · doi:10.1038/nbt.2647 · Cited by 4,741
High-Frequency off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells
Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander, J. D. (2013) · Nature Biotechnology · doi:10.1038/nbt.2623 · Cited by 3,292 · cited 1×
“Three independent studies have shownCRISPR/Cas9 could inducemutations even at the sites with up to five mismatches (Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013).”
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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
Use of Zebrafish in Drug Discovery Toxicology
Cassar, S., Adatto, I., Freeman, J. L., Gamse, J. T., Iturria, I., Lawrence, C., Muriana, A., Peterson, R. T., Van Cruchten, S., & Zon, L. I. (2020) · Chemical Research in Toxicology · doi:10.1021/acs.chemrestox.9b00335 · Cited by 561
Single-Cell Transcriptomics Meets Lineage Tracing
Kester, L., & van Oudenaarden, A. (2018) · Cell Stem Cell · doi:10.1016/j.stem.2018.04.014 · Cited by 375
A high-resolution mRNA expression time course of embryonic development in zebrafish
White, R. J., Collins, J. E., Sealy, I. M., Wali, N., Dooley, C. M., Digby, Z., Stemple, D. L., Murphy, D. N., Billis, K., Hourlier, T., et al. (2017) · eLife · doi:10.7554/elife.30860 · Cited by 361
Metadata
- Authors
- Varshney, G. K., Pei, W., LaFave, M. C., Idol, J., Xu, L., Gallardo, V., Carrington, B., Bishop, K., Jones, M., Li, M., Harper, U., Huang, S. C., Prakash, A., Chen, W., Sood, R., Ledin, J., & Burgess, S. M.
- Venue
- Genome Research
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 2015-06-05
- Volume
- 25
- Issue
- 7
- Pages
- 1030-1042
- ISSN
- 1088-9051 (ISSN-L)
- 1549-5469
- OpenAlex
- W1912701812
- Cited by
- 509
- References
- 14
- Type
- posted content
- Language
- en
- Open access
- green
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- Varshney, G. K., Pei, W., LaFave, M. C., Idol, J., Xu, L., Gallardo, V., Carrington, B., Bishop, K., Jones, M., Li, M., Harper, U., Huang, S. C., Prakash, A., Chen, W., Sood, R., Ledin, J., & Burgess, S. M. (2015). High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9. Genome Research. https://doi.org/10.1101/gr.186379.114
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