Cited by 1,046CRISPR and Genetic EngineeringRNA regulation and diseaseAnimal Genetics and Reproduction

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Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila

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Port, Fillip, Chen, Hui-Min, Lee, Tzumin, Bullock, Simon L.·2014·Proceedings of the National Academy of Sciences

DOI 10.1073/pnas.1405500111PMC PMC4115528

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Abstract

Significance: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)-mediated genome engineering promises to revolutionize genetic studies in a variety of systems. Here we describe an optimized set of tools for CRISPR/Cas experiments in the model organism Drosophila melanogaster . These tools can be used for remarkably efficient germline transmission of ( i ) loss-of-function insertion and deletion mutations in essential genes or ( ii ) precise changes in the genome sequence introduced by homology-directed repair. These tools also permit efficient biallelic targeting of genes in somatic cells, thereby demonstrating a novel application of CRISPR/Cas in rapidly revealing mutant phenotypes within the organism. Our work also paves the way for high-throughput genetic screens in Drosophila with CRISPR/Cas.

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Additional file 13 of Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development
Zhao, Y., Lindberg, B. G., Esfahani, S. S., Tang, X., Piazza, S., & Engström, Y. (2021) · Figshare · doi:10.6084/m9.figshare.16569856
Additional file 2 of Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development
Zhao, Y., Lindberg, B. G., Esfahani, S. S., Tang, X., Piazza, S., & Engström, Y. (2021) · Figshare · doi:10.6084/m9.figshare.16569874
Additional file 8 of Temporal dynamics of apoptosis-induced proliferation in pupal wing development: implications for regenerative ability
Ahmed-de-Prado, S., Estella, C., & Baonza, A. (2024) · Figshare · doi:10.6084/m9.figshare.26713741
Additional file 1 of A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila
Huynh, N., Depner, N., Larson, R. T., & King‐Jones, K. (2020) · Figshare · doi:10.6084/m9.figshare.13250775
Additional file 17 of Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development
Zhao, Y., Lindberg, B. G., Esfahani, S. S., Tang, X., Piazza, S., & Engström, Y. (2021) · Figshare · doi:10.6084/m9.figshare.16569868
Additional file 5 of Temporal dynamics of apoptosis-induced proliferation in pupal wing development: implications for regenerative ability
Ahmed-de-Prado, S., Estella, C., & Baonza, A. (2024) · Figshare · doi:10.6084/m9.figshare.26713732
Additional file 11 of Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development
Zhao, Y., Lindberg, B. G., Esfahani, S. S., Tang, X., Piazza, S., & Engström, Y. (2021) · Figshare · doi:10.6084/m9.figshare.16569850
Additional file 4 of Temporal dynamics of apoptosis-induced proliferation in pupal wing development: implications for regenerative ability
Ahmed-de-Prado, S., Estella, C., & Baonza, A. (2024) · Figshare · doi:10.6084/m9.figshare.26713729
Additional file 10 of Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development
Zhao, Y., Lindberg, B. G., Esfahani, S. S., Tang, X., Piazza, S., & Engström, Y. (2021) · Figshare · doi:10.6084/m9.figshare.16569844
Additional file 14 of Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development
Zhao, Y., Lindberg, B. G., Esfahani, S. S., Tang, X., Piazza, S., & Engström, Y. (2021) · Figshare · doi:10.6084/m9.figshare.16569859
Additional file 2 of Temporal dynamics of apoptosis-induced proliferation in pupal wing development: implications for regenerative ability
Ahmed-de-Prado, S., Estella, C., & Baonza, A. (2024) · Figshare · doi:10.6084/m9.figshare.26713723
Additional file 3 of Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development
Zhao, Y., Lindberg, B. G., Esfahani, S. S., Tang, X., Piazza, S., & Engström, Y. (2021) · Figshare · doi:10.6084/m9.figshare.16569877
Additional file 1 of Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development
Zhao, Y., Lindberg, B. G., Esfahani, S. S., Tang, X., Piazza, S., & Engström, Y. (2021) · Figshare · doi:10.6084/m9.figshare.16569871
Additional file 15 of Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development
Zhao, Y., Lindberg, B. G., Esfahani, S. S., Tang, X., Piazza, S., & Engström, Y. (2021) · Figshare · doi:10.6084/m9.figshare.16569862
Additional file 1 of Temporal dynamics of apoptosis-induced proliferation in pupal wing development: implications for regenerative ability
Ahmed-de-Prado, S., Estella, C., & Baonza, A. (2024) · Figshare · doi:10.6084/m9.figshare.26713720
Additional file 16 of Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development
Zhao, Y., Lindberg, B. G., Esfahani, S. S., Tang, X., Piazza, S., & Engström, Y. (2021) · Figshare · doi:10.6084/m9.figshare.16569865
Additional file 6 of Temporal dynamics of apoptosis-induced proliferation in pupal wing development: implications for regenerative ability
Ahmed-de-Prado, S., Estella, C., & Baonza, A. (2024) · Figshare · doi:10.6084/m9.figshare.26713735
Additional file 7 of A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila
Huynh, N., Depner, N., Larson, R. T., & King‐Jones, K. (2020) · Figshare · doi:10.6084/m9.figshare.13250793
Additional file 8 of Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development
Zhao, Y., Lindberg, B. G., Esfahani, S. S., Tang, X., Piazza, S., & Engström, Y. (2021) · Figshare · doi:10.6084/m9.figshare.16569892
Additional file 12 of Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development
Zhao, Y., Lindberg, B. G., Esfahani, S. S., Tang, X., Piazza, S., & Engström, Y. (2021) · Figshare · doi:10.6084/m9.figshare.16569853
Additional file 4 of Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development
Zhao, Y., Lindberg, B. G., Esfahani, S. S., Tang, X., Piazza, S., & Engström, Y. (2021) · Figshare · doi:10.6084/m9.figshare.16569880
Additional file 9 of Temporal dynamics of apoptosis-induced proliferation in pupal wing development: implications for regenerative ability
Ahmed-de-Prado, S., Estella, C., & Baonza, A. (2024) · Figshare · doi:10.6084/m9.figshare.26713744
Additional file 3 of Temporal dynamics of apoptosis-induced proliferation in pupal wing development: implications for regenerative ability
Ahmed-de-Prado, S., Estella, C., & Baonza, A. (2024) · Figshare · doi:10.6084/m9.figshare.26713726
Additional file 7 of Temporal dynamics of apoptosis-induced proliferation in pupal wing development: implications for regenerative ability
Ahmed-de-Prado, S., Estella, C., & Baonza, A. (2024) · Figshare · doi:10.6084/m9.figshare.26713738
Harnessing anti-CRISPR to suppress, subtract, and segregate Cas9 activity for precision CRISPR in DrosophilaPreprint
Shen, Y., Sheen, M., Yeung, A. T., Chen, X., Wang, B., Huang, Z., Ho, C. A., Lakkis, Z. I., & Han, C. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2025.12.07.692848
Evolutionary conservation of midline axon guidance activity between Drosophila and Tribolium Frazzled
Ghosh, P., Wadsworth, B. C., Terry, L., & Evans, T. A. (2025) · Developmental Biology · doi:10.1016/j.ydbio.2025.12.015
Microbiome-host interactions drive a trade-off between sleep quality and lifespan in DrosophilaPreprint
Bhargava, P., Chen, S., Lee, J., Yoshinari, Y., Nishimura, T., Ushio, M., Sugiura, Y., Cheng, K. F., Fung, Y. S., Hase, K., et al. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2025.12.24.696379
The Chimeric Nuclease SpRYc Exhibits Highly Variable Performance Across Biological Systems
Deriglazova, I. O., Shepelev, M. V., Kruglova, N. A., Georgiev, P. G., & Maksimenko, O. G. (2026) · International Journal of Molecular Sciences · doi:10.3390/ijms27010488
ExTaSy: A swappable CRISPR platform for endogenous tagging in Drosophila melanogasterPreprint
Hubbert, S., Valliji, Z., Abu-Hananah, S., Phal, S., Luchner, M., Watts, E. C., Biasi, G., Hens, K., & Kittelmann, S. (2026) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2026.01.16.699669
The Fd4 transcription factor translates transient spatial cues in progenitors into long-term lineage identityPreprint
Lai, S.-L., & Doe, C. Q. (2026) · eLife · doi:10.7554/elife.109188.2
α-Ketoglutarate couples cellular metabolism to developmental growth via hydroxylation-dependent degradation of Yorkie
He, T., Zeng, Y., Zhou, J., Tian, Y., Wang, D., Ma, J., Liu, J., Wu, W., Wei, C., & Jiao, R. (2026) · Cell Reports · doi:10.1016/j.celrep.2025.116922
Histone H3 availability is more important for development than H3.2 versus H3.3 subtype identityPreprint
McPherson, J.-M. E., Sykes, C., Grossmann, L. C., Hill, C. H., Leatham-Jensen, M. P., Duronio, R. J., & McKay, D. A. (2026) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.64898/2026.02.09.704946
Beat II and Side IV keep migrating longitudinal visceral muscle precursor cells on their substratum in Drosophila
Huang, N., Kinold, J. C., Weiß, N. W. G., Piroddi, N., Fey, I., & Aberle, H. (2026) · Journal of Cell Science · doi:10.1242/jcs.264157

Authors: Port, F., Chen, H.-M., Lee, T., & Bullock, S. L.
Affiliations: MRC Laboratory of Molecular Biology (GB)
Howard Hughes Medical Institute (US)
ORCID: Port, Fillip
Chen, Hui-Min
Lee, Tzumin
Bullock, Simon L.
Venue: Proceedings of the National Academy of Sciences
Publisher: National Academy of Sciences
Published: 2014-07-07
Volume: 111
Issue: 29
Pages: E2967-76
ISSN: 0027-8424 (ISSN-L)
1091-6490
DOI: 10.1073/pnas.1405500111
PMC: PMC4115528
OpenAlex: W1998356880
Cited by: 1,046
References: 39
Citations by year: 2026: 57 · 2025: 73 · 2024: 93 · 2023: 98 · 2022: 108 · 2021: 113 · 2020: 125 · 2019: 104 · 2018: 88 · 2017: 67 · 2016: 58 · 2015: 48 · 2014: 13
FWCI: 25.44
Type: journal article
Language: en
Open access: green
doi.org
Licence: cc-by-nc-nd
Locations: Proceedings of the National Academy of Sciences (publishedVersion)
bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
Europe PMC (PubMed Central) (publishedVersion)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
The Journal of the American Medical Association (JAMA) Network (American Medical Association) (submittedVersion, other-oa)
Funders: Howard Hughes Medical Institute
Medical Research Council (U105178790)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
Keywords: CRISPR/Cas9, Drosophila melanogaster, germline editing, somatic genome editing, homology-directed repair, loss-of-function phenotypes, high-throughput genetic screening
MeSH terms: Alleles, Animals, Base Sequence, DNA Repair, Drosophila melanogaster, Genetic Engineering, Genetic Vectors, Germ Cells, Molecular Sequence Data, Mutation, Phenotype, RNA, Genes, Insect, Gene Targeting, Transgenes, Genes, Essential, Animals, Genetically Modified, Genome, Insect, CRISPR-Cas Systems

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Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila

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